Arthropod protein disulfide isomerases

ABSTRACT

This invention relates to an isolated nucleic acid fragment encoding protein disulfide isomerase. The invention also relates to the construction of a chimeric gene encoding all or a portion of the protein disulfide isomerase, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the protein disulfide isomerase in a transformed host cell.

This application claims the benefit of U.S. Provisional Application No.60/104376, filed Oct. 15, 1998.

FIELD OF THE INVENTION

This invention is in the field of molecular biology. More specifically,this invention pertains to nucleic acid fragments encoding arthropodprotein disulfide isomerases.

BACKGROUND OF THE INVENTION

Protein folding requires the assistance of folding helpers in vivo. Theformation or isomerization of disulfide bonds in proteins is a slowprocess requiring catalysis. In nascent polypeptide chains the cysteineresidues are in the thiol form. The formation of the disulfide bondsusually occurs simultaneously with the folding of the polypeptide, inthe endoplasmic reticulum of eukaryotes or in the periplasm ofGram-negative bacteria. Cells contain three types of accessory proteinsthat function to assist polypeptides in folding to their nativeconformations: protein disulfide isomerases, propyl cis-transisomerases, and molecular chaperones.

Protein disulfide isomerase (PDI) is a homodimeric eukaryotic enzymewhich catalyzes disulfide interchange reactions. PDI is also thought tobe the beta subunit of the heterotetramer prolyl hydrolase, the enzymethat hydroxylates the proline residues in Collagen. PDI appears tobelong to a family of closely related proteins which have specificfunctions. PDI (EC 5.3.4.1), also called S-S rearrangase, catalyzes therearrangement of both intrachain and interchain disulfide bonds inproteins to form native structures. The reaction depends onsulfhydryldisulfide interchange, and PDI needs reducing agents orpartly-reduced enzyme. A family of PDI-like proteins have beenidentified in mammals, yeasts, fungi, plants, and Drosophila.

In Drosophila, a PDI precursor was identified by screening a genomic DNAlibrary at reduced stringency hybridization conditions using a ratPhospholipase C alpha cDNA probe. Northern analysis showed that thisgene encodes a transcript that is present throughout development, inheads and bodies of adults. The encoded protein contains two domainsexhibiting high similarity to thioredoxin, two regions that are similarto the hormone binding domain of human estrogen receptor, and aC-terminal ER-retention signal (KDEL). Overall, this Drosophila PDI genecontains a higher similarity to rat protein disulfide isomerase (53%identical) than to rat Phospholipase C alpha (30% identical) (McKay etal. (1995) Insect Biochem Mol Biol 25:647-654).

Another member of the PDI family is ERp-60, a PDI isoform initiallymisidentified as a phosphatidylinositol-specific phospholipase C. Thehuman and Drosophila ERp60 polypeptides have been cloned and expressed.These two ERp-60 polypeptides are similar to human PDI within almost alltheir domains, the only exception being the extreme C-terminal region.Coexpression in insect cells of the human or Drosophila ERp-60 with thealpha subunit of human propyl 4-hydrolase does not result in tetramerformation or prolyl 4-hydroxylase activity in the cells. This lack oftetramer formation is not only due to the differences in the C-terminalregion since no prolyl 4-hydroxylase tetramer is formed when a humanERp-60 hybrid containing the C-terminal region of the human PDIpolypeptide is used (Koivunen et al. (1996) Biochem J 316:599-605). The5′ flanking region of the ERp-60 gene has no TATAA box or CCAAT motifbut contains several potential binding sites for transcription factors.The highest levels of expression of the human ERp-60 mRNA are found inthe liver, placenta, lung, pancreas, and kidney, and the lowest in theheart, skeletal muscle, and brain. The ERp-60 gene has been mapped byfluorescence in situ hybridization to 15q15, a different chromosome thanwhere the human PDI and thioredoxin genes are found (Koivunen, et al.(1997) Genomics 42:397-404).

Full-length cDNA clones encoding two members of the mice PDI family havebeen cloned, sequenced, and expressed (ERp-59/PDI and ERp-72).ERp-59/PDI has been identified as the microsomal PDI. The ERp-72 aminoacid sequence shares sequence identity with ERp-59/PDI at three discreteregions, having three copies of the sequences that are thought to be theCGHC-containing active sites of ERp-59/PDI. ERp-59/PDI has the sequenceKDEL at its COOH terminus while ERp72 has the related sequence KEEL(Mazarella et al. (1990) J Biol Chem 265:1094-1101). A cDNA clonecontaining sequence similarity to the mammalian lumenal endoplasmicreticulum protein ERp-72 has been isolated from an alfalfa (Medicagosativa L.) cDNA library by screening with a cDNA encoding human PDI. Thepolypeptide encoded by this cDNA possesses a putative N-terminalsecretory signal sequence and two regions identical to the active sitesof PDI and ERp-72. This protein appears to be encoded by a small genefamily in alfalfa, whose transcripts are constitutively expressed in allmajor organs of the plant. In alfalfa cell suspension cultures, ERp-72transcripts are induced by treatment with tunicamycin, but not inresponse to calcium ionophore, heat shock or flingal elicitor (Shorroshand Dixon (1992) Plant J 2:51-58)

Another member of the PDI family is ERp-5. The amino acid sequencededuced from its cDNA insert contains two copies of the 11-amino-acidsequence Val-Glu-Phe-Tyr-Ala-Pro-Trp-Cys-Gly-His-Cys. Duplicate copiesof this sequence are found in the active sites of rat and human PDI andin Form I phosphoinositide-specific phospholipase C. Genomic sequencessimilar to the cDNA clone are amplified 10-20-fold in hamster cellsselected for resistance to increasing concentrations of hydroxyurea, aphenomenon observed earlier with cDNA clones for the M2 subunit ofribonucleotide reductase and ornithine decarboxylase. RNA blots probedwith ERp-5 cDNA show two poly(A)+ RNA species which are elevated inhydroxyurea-resistant cells (Chaudhuri et al. (1992) Biochem J281:645-650).

A PDI-like protein from Acanthamoeba castellanii contains two highlyconserved thioredeoxin-like domains, each about 100 amino acids.However, the A. castellanji PDI-like protein differs from other membersin many aspects, including the overall organization and isoelectricpoint. Southern and Northern analyses demonstrate that the PDI-likeprotein is encoded by a single-copy gene which is transcribed togenerate a 1500-nucleotide mRNA (Wong and Bateman (1994) Gene150:175-179).

Included in this application are scorpion, spider, lepidoptera, andcentepede ESTs with similarities to several of these PDIs. Coexpressionin plants or insect cells of an arthropod PDI with a secreted arthropodprotein should enhance the yield of the foreign protein by increasingthe proper folding of the foreign protein.

SUMMARY OF THE INVENTION

The present invention relates to isolated polynucleotides comprising anucleotide sequence encoding a protein disulfide isomerase precursorpolypeptide of at least 50 amino acids that has at least 80% identitybased on the Clustal method of alignment when compared to a polypeptideselected from the group consisting of a spider, a centipede, a moth, anda scorpion protein disulfide isomerase of SEQ ID NOs:2, 4, 6, and 8. Thepresent invention also relates to isolated polynucleotides comprising anucleotide sequence encoding an ERp60 polypeptide of at least 50 aminoacids that has at least 80% identity based on the Clustal method ofalignment when compared to a polypeptide selected from the groupconsisting of a spider, a centipede, and a scorpion ERp60 of SEQ IDNOs:10, 12, and 14. The present invention also relates to isolatedpolynucleotides comprising a nucleotide sequence encoding an ERp72polypeptide of at least 50 amino acids that has at least 80% identitybased on the Clustal method of alignment when compared to a polypeptideselected from the group consisting of a two scorpion ERp72s of SEQ IDNOs:16 and 18. The present invention also relates to isolatedpolynucleotides comprising a nucleotide sequence encoding an ERp5polypeptide of at least 50 amino acids that has at least 80% identitybased on the Clustal method of alignment when compared to a polypeptideselected from the group consisting of a moth, a worm, and two scorpionERp5s of SEQ ID NOs:20, 22, 24, and 26. The present invention alsorelates to an isolated polynucleotide comprising the complement of thenucleotide sequences described above.

It is preferred that the isolated polynucleotide of the claimedinvention consists of a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs:1, 3, 5, 7, 9,11, 13, 15,17,19, 21, 23, and 25that codes for the polypeptide selected from the group consisting of SEQID NOs:2, 4, 6, 8,10, 12,14, 16,18, 20, 22, 24, and 26. The presentinvention also relates to an isolated polynucleotide comprising anucleotide sequence of at least 40 (preferably at least 30) contiguousnucleotides derived from a nucleotide sequence selected from the groupconsisting of SEQ ID NOs:1, 3, 5, 7, 9,11,13,15,17,19, 21, 23, 25 andthe complement of such nucleotideqsequences.

The present invention relates to a chimeric gene comprising an isolatedpolynucleotide (such as ERp-60, an ERp-72, an ERp-5, or a PDI-likehomolog) of the present invention operably linked to suitable regulatorysequences.

The present invention relates to an isolated host cell comprising achimeric gene of the present invention or an isolated polynucleotide ofthe present invention. The host cell may be of eukaryotic origin, suchas an insect, a yeast, or a plant cell; of prokaryotic origin, such as abacterial cell. The present invention also relates to a virus,preferably a baculovirus, comprising an isolated polynucleotide of thepresent invention or a chimeric gene of the present invention.

The present invention relates to a process for producing an isolatedhost cell comprising a chimeric gene of the present invention or anisolated polynucleotide of the present invention, the process comprisingeither transforming or transfecting an isolated compatible host cellwith a chimeric gene or isolated polynucleotide of the presentinvention.

The present invention relates to a protein disulfide isomerase precursorpolypeptide of at least 30 amino acids comprising at least 80% homologybased on the Clustal method of alignment compared to a polypeptideselected from the group consisting of SEQ ID NOs:2, 4, 6, and 8. Thepresent invention relates to an ERp60 polypeptide of at least 40 aminoacids comprising at least 80% homology based on the Clustal method ofalignment compared to a polypeptide selected from the group consistingof SEQ ID NOs:10, 12, and 14. The present invention relates to an ERp72polypeptide of at least 20 amino acids comprising at least 80% homologybased on the Clustal method of alignment compared to a polypeptideselected from the group consisting of SEQ ID NOs:16 and 18. The presentinvention relates to an ERp5 polypeptide of at least 50 amino acidscomprising at least 80% homology based on the Clustal method ofalignment compared to a polypeptide selected from the group consistingof SEQ ID NOs:20, 22, 24, and 26.

The present invention relates to a method of selecting an isolatedpolynucleotide that affects the level of expression of a proteindisulfide isomerase polypeptide in a host cell, the method comprisingthe steps of:

constructing an isolated polynucleotide of the present invention or anisolated chimeric gene of the present invention;

introducing the isolated polynucleotide or the isolated chimeric geneinto a plant cell;

measuring the level an enzyme polypeptide in the plant cell containingthe isolated polynucleotide; and

comparing the level of an enzyme polypeptide in the plant cellcontaining the isolated polynucleotide or an isolated chimeric gene withthe level of an enzyme polypeptide in a plant cell that does not containthe isolated polynucleotide or an isolated chimeric gene.

presentinvention relates to a method of obtaining a nucleic acidfragment encoding a substantial portion of a protein disulfide isomerasepolypeptide gene, preferably an arthropod polypeptide gene, comprisingthe steps of: synthesizing an oligonucleotide primer comprising anucleotide sequence of at least 40 (preferably at least 30) contiguousnucleotides derived from a nucleotide sequence selected from the groupconsisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, andthe complement of such nucleotide sequences; and amplifying a nucleicacid fragment (preferably a cDNA inserted in a cloning vector) using theoligonucleotide primer. The amplified nucleic acid fragment preferablywill encode a portion of a protein disulfide isomerase amino acidsequence.

The present invention also relates to a method of obtaining a nucleicacid fragment encoding all or a substantial portion of the amino acidsequence encoding a protein disulfide isomerase polypeptide comprisingthe steps of: probing a cDNA or genomic library with an isolatedpolynucleotide of the present invention; identifying a DNA clone thathybridizes with an isolated polynucleotide of the present invention;isolating the identified DNA clone; and sequencing the cDNA or genomicfragment that comprises the isolated DNA clone.

The instant invention relates to isolated nucleic acid fragmentsencoding arthropod protein disulfide isomerases. Specifically, thisinvention concerns an isolated nucleic acid fragment encoding an ERp-60,an ERp-72, an ERp-5, or a PDI-like homolog. In addition, this inventionrelates to a nucleic acid fragment that is complementary to the nucleicacid fragment encoding ERp-60, ERp-72, ERp-5, or PDI-like homolog.

Another embodiment of the instant invention pertains to a method forexpressing a gene encoding a protein disulfide isomearase in the genomeof a recombinant baculovirus in insect cell culture or in viable insectswherein said insect cells or insects have been genetically engineered toexpress an ERp-60, an ERp-72, an ERp-5, or a PDI-like homolog.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEOUENCE LISTING

The invention can be more fully understood from the following detaileddescription and the accompanying drawings and Sequence Listing whichform a part of this application.

FIGS. 1A, 1B and 1C show a comparison of the amino acid sequences of theprotein disulfide isomerase precursors of the instant invention (SEQ IDNOs:2, 4, 6, and 8) with the sequences of protein disulfide isomeraseprecursors from Drosophila melanogaster (NCBI General IdentifierNo.1709616; SEQ ID NO:27) and Gallus gallus (NCBI General Identifier No.2144546; SEQ ID NO:28). The top row indicates with asterisks (*) theamino acids which are conserved among all the sequences from the instantinvention and one or both of the art sequences. The top row indicateswith plus signs (+) the sequences which are conserved only among thesequences from the instant invention.

FIGS. 2A, 2B, and 2C show a comparison of the amino acid sequences ofthe ERp60 of the instant invention (SEQ ID NOs:10, 12, and 14) with thesequence of ERp60 from Drosophila melanogaster(NCBI General IdentifierNo.1699220; SEQ ID NO:29). The top row indicates with asterisks (*) theamino acids which are conserved among all the sequences. The top rowindicates with plus signs (+) the sequences which are conserved onlyamong the sequences from the instant invention.

FIGS. 3A and 3B show a comparison of the amino acid sequences of theERp72 of the instant invention (SEQ ID NOs:16 and 18) with the sequenceof ERp72 from Homo sapiens (NCBI General Identifier No. 2507460; SEQ IDNO:30). The top row indicates with asterisks (*) the amino acids whichare conserved among all the sequences and with plus signs (+) thesequences which are conserved only among the arthropod sequences.

FIGS. 4A, 4B and 4C show a comparison of the amino acid sequences of theERp5 of the instant invention (SEQ ID NOs:20, 22, 24, and 26) with thesequences of ERp5 from Medicago sativa (NCBI General Identifier No.729442; SEQ ID NO:31) and Rattus norvegicus (NCBI General Identifier No.2501206; SEQ ID NO:32). The top row indicates with asterisks (*) theamino acids which are conserved among all the arthropod sequences andone or both of the art sequences. The top row indicates with plus signs(+) the sequences which are conserved only among the arthropodsequences.

Table 1 lists the polypeptides that are described herein, thedesignation of the cDNA clones that comprise the nucleic acid fragmentsencoding polypeptides representing all or a substantial portion of thesepolypeptides, and the corresponding identifier (SEQ ID NO:) as used inthe attached Sequence Listing. The sequence descriptions and SequenceListing attached hereto comply with the rules governing nucleotideand/or amino acid sequence disclosures in patent applications as setforth in 37 C.F.R. §1.821-1.825.

TABLE 1 Protein Disulfide Isomerases Clone SEQ ID NO: ProteinDesignation (Nucleotide) (Amino Acid) Spider Protein aot1c.pk016.k11  1 2 Disulfide Isomerase Precursor Centipede Protein asc1.pk033.i19  3  4Disulfide Isomerase Precursor Moth Protein Disulfide ihv1c.pk001.h12  5 6 Isomerase Precursor Scorpion Protein iks1c.pk0004.c12  7  8 DisulfideIsomerase Precursor Spider ERp-60 aot1c.pk011.n11  9 10 Scorpion ERp-60ibj1c.pk015.k8 11 12 Centipede ERp-60 isc1c.pk009.m16 13 14 ScorpionERp-72 ibj1c.pk008.d11 15 16 Scorpion ERp-72 ibj1c.pk014.c1 17 18Scorpion ERp-5 ibj1c.pk015.o22 19 20 Moth ERp-5 ihv1c.pk001.a7 21 22Scorpion ERp-5 iks1c.pk010.i14 23 24 Worm ERp-5 ise1c.pk002.m4 25 26

The Sequence Listing contains the one letter code for nucleotidesequence characters and the three letter codes for amino acids asdefined in conformity with the IUPAC-WUBMB standards described inNucleic Acids Research 13:3021-3030 (1985) and in the BiochemicalJournal 219 (No. 2):345-373 (1984) which are herein incorporated byreference. The symbols and format used for nucleotide and amino acidsequence data comply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF THE INVENTION

In the context of this disclosure, a number of terms shall be utilized.As used herein, a “polynucleotide” is a nucleotide sequence such as anucleic acid fragment. A polynucleotide may be a polymer of RNA or DNAthat is single- or double-stranded, that optionally contains synthetic,non-natural or altered nucleotide bases. A polynucleotide in the form ofa polymer of DNA may be comprised of one or more segments of cDNA,genomic DNA, or synthetic DNA. An isolated polynucleotide of the presentinvention may include at least 40 contiguous nucleotides, preferably atleast 30 contiguous nucleotides, most preferably at least 15 contiguousnucleotides, of the nucleic acid sequence of the SEQ ID NOs:1, 3, 5, 7,9, 11, 13, 15, 17, 29, 21, 23 and 25.

“NPV” stands for Nuclear Polyhedrosis Virus, a baculovirus.“Polyhedrosis” refers to any of several virus diseases of insect larvaecharacterized by dissolution of tissues and accumulation of polyhedralgranules in the resultant fluid. “PIBs” are polyhedral inclusion bodies.“AcNPV” stands for the wild-type Autographa californica NuclearPolyhedrosis Virus.

As used herein, “substantially similar” refers to nucleic acid fragmentswherein changes in one or more nucleotide bases results in substitutionof one or more amino acids, but do not affect the functional propertiesof the polypeptide encoded by the nucleotide sequence. “Substantiallysimilar” also refers to modifications of the nucleic acid fragments ofthe instant invention such as deletion or insertion of one or morenucleotides that do not substantially affect the functional propertiesof the resulting protein molecule. It is therefore understood that theinvention encompasses more than the specific exemplary nucleotide oramino acid sequences and includes functional equivalents thereof.

Substantially similar nucleic acid fragments may be selected byscreening nucleic acid fragments representing subfragments ormodifications of the nucleic acid fragments of the instant invention,wherein one or more nucleotides are substituted, deleted and/orinserted, for their ability to affect the level of the polypeptideencoded by the unmodified nucleic acid fragment in a host or host cell.For example, a substantially similar nucleic acid fragment representingat least 30 contiguous nucleotides derived from theinstant nucleic acidfragment can be constructed and introduced into a host or host cell. Thelevel of the polypeptide encoded by the unmodified nucleic acid fragmentpresent in a host or host cell exposed to the substantially similarnucleic fragment can then be compared to the level of the polypeptide ina host or host cell that is not exposed to the substantially similarnucleic acid fragment.

Consequently, an isolated polynucleotide comprising a nucleotidesequence of iat least 40 (preferably at least 30, most preferably atleast 15) contiguous nucleotides derived from a nucleotide sequenceselected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25 and the complement of such nucleotide sequencesmay be used in methods of selecting an isolated polynucleotide thataffects the expression of a polypeptide in a host cell. A method ofselecting an isolated polynucleotide that affects the level ofexpression of a polypeptide (such as a disulfide isomerase, Erp 60, Erp72, or Erp 5) in a host cell (eukaryotic, such as plant, insect, oryeast; prokaryotic such as bacterial; viral such as baculovirus) maycomprise the steps of: constructing an isolated polynucleotide of thepresent invention or an isolated chimeric gene of the present invention;introducing the isolated polynucleotide or the isolated chimeric geneinto a host cell; measuring the level of a polypeptide in the host cellcontaining the isolated polynucleotide; and comparing the level of thepolypeptide in the host cell containing the isolated polynucleotide withthe level of the polypeptide in a host cell that does not contain theisolated polynucleotide.

Moreover, substantially similar nucleic acid fragments may also becharacterized by their ability to hybridize. Estimates of such homologyare provided by either DNA-DNA or DNA-RNA hybridization under conditionsof stringency as is well understood by those skilled in the art (Hamesand Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRL Press, Oxford,U.K.). Stringency conditions can be adjusted to screen for moderatelysimilar fragments, such as homologous sequences from distantly relatedorganisms, to highly similar fragments, such as genes that duplicatefunctional enzymes from closely related organisms. Post-hybridizationwashes determine stringency conditions. One set of preferred conditionsuses a series of washes starting with 6×SSC, 0.5% SDS at roomtemperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for30 min, and then repeated twice with 2×SSC, 0.5% SDS at 50° C. for 30min. A more preferred set of stringent conditions uses ighertemperatures in which the washes are identical to those above except forthe emperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS wasincreased to 60° C. Another preferred set of highly stringent conditionsuses two final washes in 0.1×SSC, 0.1% SDS at 65° C.

Substantially similar nucleic acid fragments of the instant inventionmay also be characterized by the percent identity of the amino acidsequences that they encode to the amino acid sequences disclosed herein,as determined by algorithms commonly employed by those skilled in thisart. Suitable nucleic acid fragments (isolated polynucleotides of thepresent invention) encode polypeptides that are 80% identical to theamino acid sequences reported herein. Preferred nucleic acid fragmentsencode amino acid sequences that are 85% identical to the amino acidsequences reported herein. More preferred nucleic acid fragments encodeamino acid sequences that are 90% identical to the amino acid sequencesreported herein. Most preferred are nucleic acid fragments that encodeamino acid sequences that are 95% identical to the amino acid sequencesreported herein. Suitable nucleic acid fragments not only have the abovehomologies but typically encode a polypeptide having at least 50 aminoacids, preferably 100 amino acids, more preferably 150 amino acids,still more preferably 200 amino acids, and most preferably 250 aminoacids. Sequence alignments and percent identity calculations wereperformed using the Megalign program of the LASERGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of thesequences was performed using the Clustal method of alignment (Higginsand Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method were KTUPLE 1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5.

A “substantial portion” of an amino acid or nucleotide sequencecomprises an amino acid or a nucleotide sequence that is sufficient toafford putative identification of the protein or gene that the aminoacid or nucleotide sequence comprises. Amino acid and nucleotidesequences can be evaluated either manually by one skilled in the art, orby using computer-based sequence comparison and identification toolsthat employ algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J. Mol. Biol. 215:403-410). In general, asequence of ten or more contiguous amino acids or thirty or morecontiguous nucleotides is necessary in order to putatively identify apolypeptide or nucleic acid sequence as homologous to a known protein orgene. Moreover, with respect to nucleotide sequences, gene-specificoligonucleotide probes comprising 30 or more contiguous nucleotides maybe used in sequence-dependent methods of gene identification (e.g.,Southern hybridization) and isolation (e.g., in situ hybridization ofbacterial colonies or bacteriophage plaques). In addition, shortoligonucleotides of 12 or more nucleotides may be used as amplificationprimers in PCR in order to obtain a particular nucleic acid fragmentcomprising the primers. Accordingly, a “substantial portion” of anucleotide sequence comprises a nucleotide sequence that will affordspecific identification and/or isolation of a nucleic acid fragmentcomprising the sequence. The instant specification teaches amino acidand nucleotide sequences encoding polypeptides that comprise one or moreparticular plant proteins. The skilled artisan, having the benefit ofthe sequences as reported herein, may now use all or a substantialportion of the disclosed sequences for purposes known to those skilledin this art. Accordingly, the instant invention comprises the completesequences as reported in the accompanying Sequence Listing, as well assubstantial portions of those sequences as defined above.

“Codon degeneracy” refers to divergence in the genetic code permittingvariation of the nucleotide sequence without effecting the amino acidsequence of an encoded polypeptide. Accordingly, the instant inventionrelates to any nucleic acid fragment comprising a nucleotide sequencethat encodes all or a substantial portion of the amino acid sequencesset forth herein. The skilled artisan is well aware of the “codon-bias”exhibited by a specific host cell in usage of nucleotide codons tospecify a given amino acid. Therefore, when synthesizing a nucleic acidfragment for improved expression in a host cell, it is desirable todesign the nucleic acid fragment such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.

“Synthetic nucleic acid fragments” can be assembled from oligonucleotidebuilding blocks that are chemically synthesized using procedures knownto those skilled in the art. These building blocks are ligated andannealed to form larger nucleic acid fragments which may then beenzymatically assembled to construct the entire desired nucleic acidfragment. “Chemically synthesized”, as related to nucleic acid fragment,means that the component nucleotides were assembled in vitro. Manualchemical synthesis of nucleic acid fragments may be accomplished usingwell established procedures, or automated chemical synthesis can beperformed using one of a number of commercially available machines.Accordingly, the nucleic acid fragments can be tailored for optimal geneexpression based on optimization of nucleotide sequence to reflect thecodon bias of the host cell. The skilled artisan appreciates thelikelihood of successful gene expression if codon usage is biasedtowards those codons favored by the host. Determination of preferredcodons can be based on a survey of genes derived from the host cellwhere sequence information is available.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

“Coding sequence” refers to a nucleotide sequence that codes for aspecific amino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

“Promoter” refers to a nucleotide sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is anucleotide sequence which can stimulate promoter activity and may be aninnate element of the promoter or a heterologous element inserted toenhance the level or tissue-specificity of a promoter. Promoters may bederived in their entirety from a native gene, or be composed ofdifferent elements derived from different promoters found in nature, oreven comprise synthetic nucleotide segments. It is understood by thoseskilled in the art that different promoters may direct the expression ofa gene in different tissues or cell types, or at different stages ofdevelopment, or in response to different environmental conditions.Promoters which cause a nucleic acid fragment to be expressed in mostcell types at most times are commonly referred to as “constitutivepromoters”. New promoters of various types useful in a variety of cellsare constantly being discovered; numerous examples may be found in thecompilation by Okamuro and Goldberg (1989) Biochemistry of Plants15:1-82. It is further recognized that since in most cases the exactboundaries of regulatory sequences have not been completely defined, DNAfragments of different lengths may have identical promoter activity.

The “translation leader sequence” refers to a nucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner and Foster (1995) Mol. Biotechnol.3:225-236).

The “3′ non-coding sequences” refer to nucleotide sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′nonoding sequences is exemplified by Ingelbrecht et al. (1989) PlantCell 1:671-680.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated into proteinby the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to and derived from mRNA. “Sense” RNA refers to RNAtranscript that includes the mRNA and so can be translated into proteinby the cell. “Functional RNA” refers to sense RNA, ribozyme RNA, orother RNA that may not be translated but yet has an effect on cellularprocesses.

The term “operably linked” refers to the association of two or morenucleic acid fragments on a single nucleic acid fragment so that thefunction of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of affectingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in sense orientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression may also refer totranslation of mRNA into a polypeptide. “Overexpression” refers to theproduction of a gene product in transgenic organisms that exceeds levelsof production in normal or non-transformed organisms.

“Altered levels” refers to the production of gene product(s) intransgenic organisms in amounts or proportions that differ from that ofnormal or non-transformed organisms.

A “signal sequence” is an amino acid sequence that is covalently linkedto an amino acid sequence representing a mature protein. The signalsequence directs the protein to the secretory system (Chrispeels (1991)Ann Rev. Plant Phys. Plant Mol. Biol. 42:21-53). “Mature” protein refersto a post-translationauy processed polypeptide; i.e., one from which anypre- or propeptides, including signal sequences, present in the primarytranslation product have been removed. “Precursor” protein refers to theprimary product of translation of mRNA; i.e., with pre- and propeptidesstill present. Pre- and propeptides may be but are not limited tointracellular localization signals.

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. Examples of methodsof plant transformation include Agrobacterium-mediated transformation(De Blaere et al. (1987) Meth. Enzymol. 143:277) andparticle-accelerated or “gene gun” transformation technology (Klein etal. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050,incorporated herein by reference).

It is understood that “an insect cell” refers to one or more insectcells maintained in vitro as well as one or more cells found in anintact, living insect.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook etal. Molecular Cloning: A Laboratory Manual; Cold Spring HarborLaboratory Press: Cold Spring Harbor, 1989 (hereinafter “Maniatis”).

Nucleic acid fragments encoding at least a portion of several proteindisulfide isomerases have been isolated and identified by comparison ofcDNA sequences to public databases containing nucleotide and proteinsequences using the BLAST algorithms well known to those skilled in theart. The nucleic acid fragments of the instant invention may be used toisolate cDNAs and genes encoding homologous proteins from the same orother arthropod species. Isolation of homologous genes usingsequence-dependent protocols is well known in the art. Exanples ofsequence-dependent protocols include, but are not limited to, methods ofnucleic acid hybridization, and methods of DNA and RNA amplification asexemplified by various uses of nucleic acid amplification technologies(e.g., polymerase chain reaction, ligase chain reaction).

For example, genes encoding other ERp-60s, ERp-72s, ERp-5s, or PDI-likehomologs, either as cDNAs or genomic DNAs, could be isolated directly byusing all or a portion of the instant nucleic acid fragments as DNAhybridization probes to screen libraries from any desired arthropodemploying methodology well known to those skilled in the art. Specificoligonucleotide probes based upon the instant nucleic acid sequences canbe designed and synthesized by methods known in the art (Maniatis).Moreover, the entire sequences can be used directly to synthesize DNAprobes by methods known to the skilled artisan such as random primer DNAlabeling, nick translation, or end-labeling techniques, or RNA probesusing available in vitro transcription systems. In addition, specificprimers can be designed and used to amplify a part or all of the instantsequences. The resulting amplification products can be labeled directlyduring amplification reactions or labeled after amplification reactions,and used as probes to isolate full length cDNA or genomic fragmentsunder conditions of appropriate stringency.

In addition, two short segments of the instant nucleic acid fragmentsmay be used in polymerase chain reaction protocols to amplify longernucleic acid fragments encoding homologous genes from DNA or RNA. Thepolymerase chain reaction may also be performed on a library of clonednucleic acid fragments wherein the sequence of one primer is derivedfrom the instant nucleic acid fragments, and the sequence of the otherprimer takes advantage of the presence of the polyadenylic acid tractsto the 3′ end of the mRNA precursor encoding arthropod genes.Alternatively, the second primer sequence may be based upon sequencesderived from the cloning vector. For example, the skilled artisan canfollow the RACE protocol (Frohman et al. (1988) Proc. Natl. Acad. Sci.USA 85:8998-9002) to generate cDNAs by using PCR to amplify copies ofthe region between a single point in the transcript and the 3′ or 5′end. Primers oriented in the 3′ and 5′ directions can be designed fromthe instant sequences. Using commercially available 3′ RACE or 5′ RACEsystems (BRL), specific 3′ or 5′ cDNA fragments can be isolated (Oharaet al. (1989) Proc. Natl. Acad. Sci. USA 86:5673-5677; Loh et al. (1989)Science 243:217-220). Products generated by the 3′ and 5′ RACEprocedures can be combined to generate full-length cDNAs (Frohman andMartin (1989) Techniques 1:165). Consequently, a polynucleotidecomprising a nucleotide sequence of at least 40 (preferably one of atleast 30, most preferably one of at least 15) contiguous nucleotidesderived from a nucleotide sequence selected from the group consisting ofSEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 and thecomplement of such nucleotide sequences may be used in such methods toobtain a nucleic acid fragment encoding a substantial portion of anamino acid sequence of a polypeptide such as a protein disulfideisomerase. The present invention relates to a method of obtaining anucleic acid fragment encoding a substantial portion of a polypeptide ofa gene (such as ERp-60, an ERp-72, an ERp-5, or a PDI-like homolog)preferably a substantial portion of an arthropod polypeptide of a gene,comprising the steps of: synthesizing an oligonucleotide primercomprising a nucleotide sequence of at least 40 (preferably at least 30,most preferably at least 15) contiguous nucleotides derived from anucleotide sequence selected from the group consisting of SEQ ID NOs:1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 and the complement of suchnucleotide sequences; and amplifying a nucleic acid fragment (preferablya cDNA inserted in a cloning vector) using the oligonucleotide primer.The amplified nucleic acid fragment preferably will encode a portion ofa polypeptide such as a protein disulfide isomerase.

Availability of the instant nucleotide and deduced amino acid sequencesfacilitates immunological screening of cDNA expression libraries.Synthetic peptides representing portions of the instant amino acidsequences may be synthesized. These peptides can be used to immunizeanimals to produce polyclonal or monoclonal antibodies with specificityfor peptides or proteins comprising the amino acid sequences. Theseantibodies can be then be used to screen cDNA expression libraries toisolate full-length cDNA clones of interest (Lemer (1984) Adv. Immunol.36:1-34; Maniatis).

The nucleic acid fragments of the instant invention may be used tocreate transgenic plants in which the disclosed protein disulfideisomerases are expressed. This would be useful as a means forcontrolling insect pests by producing plants that are moreinsect-tolerant than the naturally occurring variety.

Expression in plants of the proteins of the instant invention may beaccomplished by first constructing a chimeric gene in which the codingregion is operably linked to a promoter capable of directing expressionof a gene in the desired tissues at the desired stage of development.For reasons of convenience, the chimeric gene may comprise promotersequences and translation leader sequences derived from the same genes.3′ Non-coding sequences encoding transcription termination signals mayalso be provided. The instant chimeric gene may also comprise one ormore introns in order to facilitate gene expression.

Plasmid vectors comprising the instant chimeric gene can thenconstructed. The choice of plasmid vector is dependent upon the methodthat will be used to transform host plants. The skilled artisan is wellaware of the genetic elements that must be present on the plasmid vectorin order to successfully transform, select and propagate host cellscontaining the chimeric gene. The skilled artisan will also recognizethat different independent transformation events will result indifferent levels and patterns of expression (Jones et al. (1985) EMBO J.4:2411-2418; De Almeida et al. (1989) Mol. Gen. Genetics 218:78-86), andthus that multiple events must be screened in order to obtain linesdisplaying the desired expression level and pattern. Such screening maybe accomplished by Southern analysis of DNA, Northern analysis of mRNAexpression, Western analysis of protein expression, LC-MS, or phenotypicanalysis.

The instant polypeptides (or portions thereof) may be produced inheterologous host cells, particularly in the cells of microbial hosts,and can be used to prepare antibodies to the these proteins by methodswell known to those skilled in the art. The antibodies are useful fordetecting the polypeptides of the instant invention in situ in cells orin vitro in cell extracts. Preferred heterologous host cells forproduction of the instant polypeptides are microbial hosts. Microbialexpression systems and expression vectors containing regulatorysequences that direct high level expression of foreign proteins are wellknown to those skilled in the art. Any of these could be used toconstruct a chimeric gene for production of the instant polypeptides.This chimeric gene could then be introduced into appropriatemicroorganisms via transformation to provide high level expression ofthe encoded protein disulfide isomerase. An example of a vector for highlevel expression of the instant polypeptides in a bacterial host isprovided (Example 9).

Insecticidal baculoviruses have great potential to provide anenvironmentally benign method for agricultural insect pest control.However, improvements to efficacy are required in order to make theseagents competitive with current chemical pest control agents. Oneapproach for making such improvements is through genetic alteration ofthe virus. For instance, it may be possible to modify the viral genomein order to improve the host range of the virus, to increase theenvironmental stability and persistence of the virus, or to improve theinfectivity and transmission of the virus. In addition, improving therate at which the virus acts to compromise the infected insect wouldsignificantly enhance the attractiveness of insecticidal baculovirusesas adjuncts or replacements for chemical pest control agents. One methodfor increasing the speed with which the virus affects its insect host isto introduce into the baculovirus foreign genes that encode proteinsthat are toxic to the insect wherein death or incapacitation of theinsect is no longer dependent solely on the course of the viralinfection, but instead is aided by the accumulation of toxic levels ofthe foreign protein. The results are insecticidal recombinantbaculoviruses.

Recombinant baculoviruses expressing the instant protein disulfideisomerase (or portions thereof) may be prepared by protocols now knownto the art (e.g., Tomalski et al., U.S. Pat. No. 5,266,317, exemplifingneurotoxins from the insect-parasitic mites; McCutchen et al. (1991)Bio/Technology 9:848-852; Maeda et al. (1991) Virology 184:777-780,illustrating construction of a recombinant baculovirus expressing AaIT;also see O'Reilly et al. (1992) Baculovirus Expression Vectors: ALaboratory Manual, W. H. Freeman and Company, New York; King and Possee(1992) The Baculovirus Expression System, Chapman and Hall, London; U.S.Pat. No. 4,745,051). These methods of gene expression provide economicalpreparation of foreign proteins in a eukaryotic expression vectorsystem, in many instances yielding proteins that have achieved theirproper tertiary conformation and formed the proper disulfide bridgesnecessary for activity.

Commonly, the introduction of heterologous genes into the baculovirusgenome occurs by homologous recombination between viral genomic DNA anda suitable “transfer vector” containing the heterologous gene ofinterest. These transfer vectors are generally plasmid DNAs that arecapable of autonomous replication in bacterial hosts, affording facilegenetic manipulation. Baculovirus transfer vectors also contain agenetic cassette comprising a region of the viral genome that has beenmodified to include the following features (listed in the 5′ to 3′direction): 1) viral DNA comprising the 5′ region of a non-essentialgenomic region; 2) a viral promoter; 3) one or more DNA sequencesencoding restriction enzyme sites facilitating insertion of heterologousDNA sequences; 4) a transcriptional termination sequence; and 5) viralDNA comprising the 3′ region of a non-essential genomic region. Aheterologous gene of interest is inserted into the transfer vector atthe restriction site downstream of the viral promoter. The resultingcassette comprises a chimeric gene wherein the heterologous gene isunder the transcriptional control of the viral promoter andtranscription termination sequences present on the transfer vector.Moreover, this chimeric gene is flanked by viral DNA sequences thatfacilitate homologous recombination at a non-essential region of theviral genome. Recombinant viruses are created by co-transfecting insectcells that are capable of supporting viral replication with viralgenomic DNA and the recombinant transfer vector. Homologousrecombination between the flanking viral DNA sequences present on thetransfer vector and the homologous sequences on the viral genomic DNAtakes place and results in insertion of the chimeric gene into a regionof the viral genome that does not disrupt an essential viral function.The infectious recombinant virion consists of the recombined genomicDNA, referred to as the baculovirus expression vector, surrounded by aprotein coat.

In a preferred embodiment, the non-essential region of the viral genomethat is present on the transfer vector comprises the region of the viralDNA responsible for polyhedrin production. Most preferred is a transfervector that contains the entire polyhedrin gene between the flankingsequences that are involved in homologous recombination. Recombinationwith genomic DNA from viruses that are defective in polyhedrinproduction (due to a defect in the genomic copy of the polyhedrin gene)will result in restoration of the polyhedrin-positive phenotype. Thisstrategy facilitates identification and selection of recombinantviruses.

In another embodiment, baculoviral genomic DNA can be directly modifiedby introduction of a unique restriction enzyme recognition sequence intoa non-essential region of the viral genome. A chimeric gene comprisingthe heterologous gene to be expressed by the recombinant virus andoperably linked to regulatory sequences capable of directing geneexpression in baculovirus-infected insect cells, can be constructed andinserted directly into the viral genome at the unique restriction site.This strategy eliminates both the need for construction of transfervectors and reliance on homologous recombination for generation ofrecombinant viruses. This technology is described by Ernst et al. (Ernstet al. (1994) Nuc. Acid Res. 22: 2855-2856), and in WO 94/28114.

Recombinant baculovirus expression vectors suitable for deliveringgenetically encoded insect-specific protein disulfide isomerases requireoptimal gene expression for maximum efficacy. A number of strategies canbe used by the skilled artisan to design and prepare recombinantbaculoviruses wherein protein disulfide isomerase gene expressionresults in sufficient quantities of protein disulfide isomerase producedat appropriate times during infection in a functional form and availablefor binding to target cells within the insect host.

The isolated protein disulfide isomerase gene fragment may be digestedwith appropriate enzymes and may be inserted into the pTZ-18R plasmid(Pharmacia, Piscataway, N.J.) at the multiple cloning site usingstandard molecular cloning techniques. Following transformation of E.coli DH5αMCR, isolated colonies may be chosen and plasmid DNA prepared.Positive clones will be identified and sequenced with the commerciallyavailable forward and reverse primers.

Spodopteraftugiperda cells (Sf-9) may be propagated in ExCell®401 media(JRH Biosciences, Lenexa, Kans.) supplemented with 3.0% fetal bovineserum. Lipofectin® (50 μL at 0.1 mg/mL, Gibco/BRL) may be added to a 50μL aliquot of the transfer vector containing the toxin gene of interest(500 ng) and linearized polyhedrin-negative AcNPV (2.5 μg, BaculogoldDviral DNA, Pharmigen, San Diego, Calif.). Sf-9 cells (approximate 50%monolayer) may be co-transfected with the viral DNA/transfer vectorsolution. The supernatant fluid from the co-transfection experiment maybe collected at 5 days post-transfection and recombinant viruses may beisolated employing standard plaque purification protocols, wherein onlypolyhedrin-positive plaques will be selected (Granados and Lawler (1981)Virology 108:297-308).

To propagate the recombinant virus of interest, isolated plaques may bepicked and suspended in 500 μL of ExCell® media supplemented with 2.5%fetal bovine serum. Sf-9 cells in 35 mM petri dishes (50% monolayer) maybe inoculated with 100 μL of the viral suspension, and supernatantfluids collected at 5 days post infection. These supernatant fluids willbe used to inoculate cultures for large scale propagation of recombinantviruses.

Expression of the encoded protein disulfide isomerase gene by therecombinant baculovirus will be confirmed using a bioassay, LCMS, orantibodies.

EXAMPLES

The present invention is further defined in the following Examples, inwhich all parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usages and conditions.

Example 1

Composition of cDNA Libraries: Isolation and Sequencing of cDNA Clones

cDNA libraries representing mRNAs from various spider, caterpillar,centipede, moth, and scorpion tissues were prepared. The characteristicsof the libraries are described below.

TABLE 2 cDNA Libraries from Scorpion, Caterpillar, Centipede, Moth, andSpider Library Tissue Clone aot1c Ceplalothorax of orb spider collectedfrom aot1c.pk011.n11 Texas aot1c Ceplalothorax of orb spider collectedfrom aot1c.pk016.k11 Texas asc1 Scolopendra canidens DS milked venomasc1.pk033.i19 glands ibj1c Buthatus judaicus Telsons 48-Hours Postibj1c.pk008.d11 Milking ibj1c Buthatus judaicus Telsons 48-Hours Postibj1c.pk015.k8 Milking ibj1c Buthatus judaicus Telsons 48-Hours Postibj1c.pk015.o22 Milking ibj1c Buthatus judaicus Telsons 48-Hours Postibj1c.pk014.c1 Milking ihv1c Tobacco budworm (Heliothis virescens)ihv1c.pk001.a7 Whole Insect ihv1c Tobacco budworm (Heliothis virescens)ihv1c.pk001.h12 Whole Insect iks1c Kentucky scorpion Telsons 48-HoursPost iks1c.pk0004.c12 Milking iks1c Kentucky scorpion Telsons 48-HoursPost iks1c.pk010.i14 Milking isc1c Scolopendra canidens Telsons 48-HoursPost isc1c.pk009.m16 Milking isc1c Fall armyworm (Spodoptera exigua)ise1c.pk002.m4 Whole Insect

cDNA libraries may be prepared by any one of many methods available. Forexample, the cDNAs may be introduced into plasmid vectors by firstpreparing the cDNA libraries in Unc-ZAP™ XR vectors according to themanufacturer's protocol (Stratagene Cloning Systems, La Jolla, Calif.).The Uni-ZAP™ XR libraries are converted into plasmid libraries accordingto the protocol provided by Stratagene. Upon conversion, cDNA insertswill be contained in the plasmid vector pBluescript. In addition, thecDNAs may be introduced directly into precut Bluescript II SK(+) vectors(Stratagene) using T4 DNA ligase (New Eng land Biolabs), followed bytransfection into DH10B cells according to the manufacturer's protocol(GIBCO BRL Products). Once the cDNA inserts are in plasmid vectors,plasmid DNAs are prepared from randomly picked bacterial coloniescontaining recombinant pbluescript plasmids, or the insert eDNAsequences are amplified via polymerase chain reaction using primersspecific for vector sequences flanking the inserted cDNA sequences.Amplified insert DNAs or plasmid DNAs are sequenced in dye-primersequencing reactions to generate partial cDNA sequences (expressedsequence tags or “ESTs”; see Adams et al., (1991) Science252:1651-1656). The resulting ESTs are analyzed using a Perkin ElmerModel 377 fluorescent sequencer.

Example 2 Identification of cDNA Clones

ESTs encoding protein disulfide isomerases were identified by conductingBLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol.Biol. 215:403-410) searches for similarity to sequences contained in theBLAST “nr” database (comprising all non-redundant GenBank CDStranslations, sequences derived from the 3-dimensional structureBrookhaven Protein Data Bank, the last major release of the SWISS-PROTprotein sequence database, EMBL, and DDBJ databases). The cDNA sequencesobtained in Example 1 were analyzed for similarity to all publiclyavailable DNA sequences contained in the “nr” database using the BLASTNalgorithm provided by the National Center for Biotechnology Information(NCBI). The DNA sequences were translated in all reading frames andcompared for similarity to all publicly available protein sequencescontained in the “nr” database using the BLASTX algorithm (Gish andStates (1993) Nat. Genet. 3:266-272) provided by the NCBI. Forconvenience, the P-value (probability) of observing a match of a cDNAsequence to a sequence contained in the searched databases merely bychance as calculated by BLAST are reported herein as “pLog” values,which represent the negative of the logarithm of the reported P-value.Accordingly, the greater the pLog value, the greater the likelihood thatthe cDNA sequence and the BLAST “hit” represent homologous proteins.

Example 3 Characterization of cDNA Clones Encoding Protein DisulfideIsomerase Precursor

The BLASTX search using the EST sequences from clones listed in Table 3revealed similarity of the polypeptides encoded by the cDNAs to proteindisulfide isomerase precursor from Drosophila melanogaster or Gallusgallus (NCBI General Identifier Nos. 4262594, 1709616, and 2144546,respectively). Shown in Table 3 are the BLAST results for individualESTs (“EST”), or the sequences of the entire cDNA inserts comprising theindicated cDNA clones (“FIS”):

TABLE 3 BLAST Results for Sequences Encoding Polypeptides Homologous toProtein Disulfide Isomerase Precursor NCBI General Clone StatusIdentifier No. BLAST pLog Score aot1c.pk016.k11 FIS 1709616 166.00asc1.pk033.i19 FIS 2144546 168.00 ihv1c.pk001.h12 EST 1709616  56.00iks1c.pk0004.c12 EST 2144546 165.00

The data in Table 4 represents a calculation of the percent identity ofthe amino acid sequences set forth in SEQ ID NOs:2, 4, 6, and 8 and theDrosophila melanogaster and Gallus gallus sequences (NCBI GeneralIdentifier Nos. 1709616 and 2144546, respectively).

TABLE 4 Percent Identity of Amino Acid Sequences Deduced From theNucleo- tide Sequences of cDNA Clones Encoding Polypeptides Homologousto Protein Disulfide Isomerase Precursor Percent Identity to SEQ ID NO.1709616 2144546 2 54.8 53.8 4 54.6 55.8 6 65.4 60.2 8 53.2 54.1

Sequence alignments and percent identity calculations were performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequenceswas performed using the Clustal method of alignment (Higgins and Sharp(1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=10). Default parameters for pairwise alignments usingthe Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5. Sequence alignments and BLAST scores and probabilities indicatethat the nucleic acid fragments comprising the instant cDNA clonesencode a substantial portion of a spider, a centipede, a moth and ascorpion PDI precursor. These sequences represent the first arthropodsequences encoding protein disulfide isomerase precursor.

Example 4 Characterization of cDNA Clones Encoding ERp-60

The BLASTX search using the EST sequences from clones listed in Table 5revealed similarity of the polypeptides encoded by the cDNAs to ERp-60from Drosophila melanogaster (NCBI General Identifier No. 1699220).Shown in Table 5 are the BLAST results for the sequences of the entirecDNA inserts comprising the indicated cDNA clones (“FIS”):

TABLE 5 BLAST Results for Sequences Encoding Polypeptides Homologous toERp-60 BLAST pLog Score Clone Status 1699220 aot1c.pk011.n11 FIS 170.00ibj1c.pk015.k8 FIS 153.00 isc1c.pk009.m16 FIS 172.00

The data in Table 6 represents a calculation of the percent identity ofthe amino acid sequences set forth in SEQ ID NOs:10, 12 and 14 and theDrosophila melanogaster sequence (1699220).

TABLE 6 Percent Identity of Amino Acid Sequences Deduced From theNucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous toERp-60 Percent Identity to SEQ ID NO. 1699220 10 56.2 12 55.4 14 55.3

Sequence alignments and percent identity calculations were performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequenceswas performed using the Clustal method of alignment (Higgins and Sharp(1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=10). Default parameters for pairwise alignments usingthe Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5. Sequence alignments and BLAST scores and probabilities indicatethat the nucleic acid fragments comprising the instant cDNA clonesencode a substantial portion of a spider, a scorpion, and a centipedeERp-60. These sequences represent the first arthropod sequences encodingERp-60.

Example 5 Characterization of cDNA Clones Encoding ERp-72

The BLASTX search using the EST sequences from clones listed in Table 7revealed similarity of the polypeptides encoded by the cDNAs to ERp-72from Homo sapiens (NCBI General Identifier No. 2507460). Shown in Table7 are the BLAST results for the sequences of the entire cDNA insertscomprising the indicated cDNA clones (“FIS”):

TABLE 7 BLAST Results for Sequences Encoding Polypeptides Homologous toERp-72 BLAST pLog Score Clone Status 2507460 ibj1c.pk005.d11 FIS 161.00ibj1c.pk014.c1 FIS 160.00

The data in Table 8 represents a calculation of the percent identity ofthe amino acid sequences set forth in SEQ ID NOs:16 and 18 and the Homosapiens sequence (NCBI General Identifier No. 2507460).

TABLE 8 Percent Identity of Amino Acid Sequences Deduced From theNucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous toERp-72 Percent Identity to SEQ ID NO. 2507460 16 53.8 18 53.6

Sequence alignments and percent identity calculations were performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequenceswas performed using the Clustal method of alignment (Higgins and Sharp(1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=10). Default parameters for pairwise alignments usingthe Clustal method were KTUPLE 1, GAP PENALTY=3, UtNOW=5 and DIAGONALSSAVED=5. Sequence alignments and BLAST scores and probabilities indicatethat the nucleic acid fragments comprising the instant cDNA clonesencode two entire scorpion ERp-72. These sequences represent the firstscorpion sequences encoding ERp-72.

Example 6 Characterization of cDNA Clones Encoding, ERD-5

The BLASTX search using the EST sequences from clones listed in Table 9revealed similarity of the polypeptides encoded by the cDNAs to ERp-5from Medicago saliva or Rattus norvegicus (NCBI General Identifier Nos.729442 and 2501206, respectively). Shown in Table 9 are the BLASTresults for individual ESTs (“EST”), or the sequences of the entire cDNAinserts comprising the indicated cDNA clones (“FIS”):

TABLE 9 BLAST Results for Sequences Encoding Polypeptides Homologous toERp-5 NCBI General Clone Status Identifier No. BLAST pLog Scoreibj1c.pk015.o22 FIS 729442   38.00 ihv1c.pk001.a7 FIS 2501206 105.00iks1c.pk010.i14 EST 729442   39.15 ise1c.pk002.m4 FIS 2501206 147.00

The data in Table 10 represents a calculation of the percent identity ofthe amino acid sequences set forth in SEQ ID NOs:18, 20, 22, and 24 andthe Medicago saliva and Rattus norvegicus sequences (NCBI GeneralIdentifier Nos. 729442 and 2501206, respectively).

TABLE 10 Percent Identity of Amino Acid Sequences Deduced From theNucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous toERp-5 Percent Identity to SEQ ID NO. 729442 2501206 18 27.9 22.5 20 19.459.2 22 24.2 19.2 24 26.4 55.6

Sequence alignments and percent identity calculations were performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequenceswas performed using the Clustal method of alignment (Higgins and Sharp(1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=10). Default parameters for pairwise alignments usingthe Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5. Sequence alignments and BLAST scores and probabilities indicatethat the nucleic acid fragments comprising the instant cDNA clonesencode a substantial portion of a caterpillar, a moth, and two scorpionERp-5. These sequences represent the first arthropod sequences encodingERp-5.

Example 7 Expression of Chimeric Genes in Monocot Cells

A chimeric gene comprising a cDNA encoding the instant polypeptides insense orientation with respect to the maize 27 kD zein promoter that islocated 5′ to the cDNA fragment, and the 10 kD zein 3′ end that islocated 3′ to the cDNA fragment, can be constructed. The cDNA fragmentof this gene may be generated by polymerase chain reaction (PCR) of thecDNA clone using appropriate oligonucleotide primers. Cloning sites (NcoI or Sma I) can be incorporated into the oligonucleotides to provideproper orientation of the DNA fragment when inserted into the digestedvector pML103 as described below. Amplification is then performed in astandard PCR. The amplified DNA is then digested with restrictionenzymes Nco I and Sma I and fractionated on an agarose gel. Theappropriate band can be isolated from the gel and combined with a 4.9 kbNco I-Sma I fragment of the plasmid pML103. Plasmid pML103 has beendeposited under the terms of the Budapest Treaty at ATCC (American TypeCulture Collection, 10801 University Blvd., Manassas, Va. 20110-2209),and bears accession number ATCC 97366. The DNA segment from pML103contains a 1.05 kb Sal I-Nco I promoter fragment of the maize 27 kD zeingene and a 0.96 kb Sma I-Sal I fragment from the 3′ end of the maize 10kD zein gene in the vector pGem9Zf(+) (Promega). Vector and insert DNAcan be ligated at 15° C. overnight, essentially as described (Maniatis).The ligated DNA may then be used to transform E. coli XL1-Blue(Epicurian Coli XL-1 Blue™; Stratagene). Bacterial transformants can bescreened by restriction enzyme digestion of plasmid DNA and limitednucleotide sequence analysis using the dideoxy chain termination method(Sequenase™ DNA Sequencing Kit; U.S. Biochemical). The resulting plasmidconstruct would comprise a chimeric gene encoding, in the 5′ to 3′direction, the maize 27 kD zein promoter, a cDNA fragment encoding theinstant polypeptides, and the 10 kD zein 3′ region.

The chimeric gene described above can then be introduced into corn cellsby the following procedure. Immature corn embryos can be dissected fromdeveloping caryopses derived from crosses of the inbred corn lines H99and LH132. The embryos are isolated 10 to 11 days after pollination whenthey are 1.0 to 1.5 mm long. The embryos are then placed with theaxis-side facing down and in contact with agarose-solidified N6 medium(Chu et al., (1975) Sci. Sin. Peking 18:659-668). The embryos are keptin the dark at 27° C. Friable embryogenic callus consisting ofundifferentiated masses of cells with somatic proembryoids and embryoidsborne on suspensor structures proliferates from the scutellum of theseimmature embryos. The embryogenic callus isolated from the primaryexplant can be cultured on N6 medium and sub-cultured on this mediumevery 2 to 3 weeks.

The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag,Frankfurt, Germany) may be used in transformation experiments in orderto provide for a selectable marker. This plasmid contains the Pat gene(see European Patent Publication 0 242 236) which encodesphosphinothricin acetyl transferase (PAT). The enzyme PAT confersresistance to herbicidal glutamine synthetase inhibitors such asphosphinothricin. The pat gene in p35S/Ac is under the control of the35S promoter from cauliflower mosaic virus (Odell et al. (1985) Nature313:810-812) and the 3′ region of the nopaline synthase gene from theT-DNA of the Ti plasmid of Agrobacterium tumefaciens.

The particle bombardment method (Klein et al. (1987) Nature 327:70-73)may be used to transfer genes to the callus culture cells. According tothis method, gold particles (1 μm in diameter) are coated with DNA usingthe following technique. Ten μg of plasmid DNAs are added to 50 μL of asuspension of gold particles (60 mg per mL). Calcium chloride (50 μL ofa 2.5 M solution) and spermidine free base (20 μL of a 1.0 M solution)are added to the particles. The suspension is vortexed during theaddition of these solutions. After 10 minutes, the tubes are brieflycentrifuged (5 sec at 15,000 rpm) and the supernatant removed. Theparticles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles can be placed in thecenter of a Kapton™ flying disc (Bio-Rad Labs). The particles are thenaccelerated into the corn tissue with a Biolistic™ PDS-1000/He (Bio-RadInstruments, Hercules Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 cm and a flying distance of 1.0 cm.

For bombardment, the embryogenic tissue is placed on filter paper overagarose-solidified N6 medium. The tissue is arranged as a thin lawn andcovered a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

Seven days after bombardment the tissue can be transferred to N6 mediumthat contains gluphosinate (2 mg per liter) and lacks casein or proline.The tissue continues to grow slowly on this medium. After an additional2 weeks the tissue can be transferred to fresh N6 medium containinggluphosinate. After 6 weeks, areas of about 1 cm in diameter of activelygrowing callus can be identified on some of the plates containing theglufosinate-supplemented medium. These calli may continue to grow whensubcultured on the selective medium.

Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Frommn et al., (1990) Bio/Technology 8:833-839).

Example 8 Expression of Chimeric Genes in Dicot Cells

A seed-specific expression cassette composed of the promoter andtrncription terminator from the gene encoding the P subunit of the seedstorage protein phaseolin from the bean Phaseolus vulgaris (Doyle et al.(1986) J. Biol. Chem. 261:9228-9238) can be used for expression of theinstant polypeptides in transformed soybean. The phaseolin cassetteincludes about 500 nucleotides upstream (5′) from the translatioeinitiation codon about 1650 nucleotides downstream (3′) from thetranslation stop codon of phaseolin. Between the 5′ and 3′ regions arethe unique restriction endonuclease sites Nco I (which includes the ATOtranslation initiation codon), Sma I, Kpn I and Xba I. The entirecassette is flanked by Hind Ill sites.

The cDNA fragment of this gene may be generated by polymerase chainreaction (P)CR) of the cDNA clone using appropriate oligonucleotideprimers. Cloning sites can be incorporated into the oligonucleotides toprovide proper orientation of the DNA fragment when inserted into theexpression vector. Amplification is then performed as described above,and the isolated fragment is inserted into a pVC 1 8 vector carrying theseed expression cassette.

Soybean embryos may then be transformed with the expression vectorcomprising sequences encoding the instant polypeptides. To inducesomatic embryos, cotyledons, 3-5 mm in length dissected from surfacesterilized, immature seeds of the soybean cultivar A2872, can becultured in the light or dark at 26° C. on an appropriate agar mediumfor 6-10 weeks. Somatic embryos which produce secondary embryos are thenexcised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos which multiplied as early,globular staged embryos, the suspensions are maintained as describedbelow.

Soybean embryogenic suspension cultures can maintained in 35 mL liquidmedia on a rotary shaker, 150 rpm, at 26° C. with florescent lights on a16:8 hour day/night schedule. Cultures are subcultured every two weeksby inoculating approximately 35 mg of tissue into 35 mL of liquidmedium. Soybean embryogenic suspension cultures may then be transformedby the method of particle gun bombardment (Klein et al. (1987) Nature(London) 32 7:70, U.S. Pat. No. 4,945,050). A DuPont BiolisticT™PDS1000/HE instrument (helium retrofit) can be used for thesetransformations.

A selectable marker gene which can be used to facilitate soybeantransformation is a chimeric gene composed of the 35S promoter fromcauliflower mosaic virus (Odell et al. (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al.(1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The seed expression cassette comprising the phaseolin 5′region, the fragment encoding the instant polypeptides and the phaseolin3′ region can be isolated as a restriction fragment. This fragment canthen be inserted into a unique restriction site of the vector carryingthe marker gene.

To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five μL of theDNA-coated gold particles are then loaded on each macro carrier disk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi and the chamber is evacuated to a vacuum of 28 inchesmercury. The tissue is placed approximately 3.5 inches away from theretaining screen and bombarded three times. Following bombardment, thetissue can be divided in half and placed back into liquid and culturedas described above.

Five to seven days post bombardment, the liquid media may be exchangedwith fresh media, and eleven to twelve days post bombardment with freshmedia containing 50 mg/nL hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 9 Expression of Chimeric Genes in Microbial Cells

The cDNAs encoding the instant polypeptides can be inserted into the T7E. coli expression vector pBT430. This vector is a derivative of pET-3a(Rosenberg et al. (1987) Gene 56:125-135) which employs thebacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 wasconstructed by first destroying the EcoR I and Hind III sites in pET-3aat their original positions. An oligonucleotide adaptor containing EcoRI and Hind III sites was inserted at the BamH I site of pET-3a. Thiscreated pET-3aM with additional unique cloning sites for insertion ofgenes into the expression vector. Then, the Nde I site at the positionof translation initiation was converted to an Nco I site usingoligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM inthis region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

Plasmid DNA containing a cDNA may be appropriately digested to release anucleic acid fragment encoding the protein. This fragment may then bepurified on a 1% NuSieve GTG™ low melting agarose gel (FMC). Buffer andagarose contain 10 μg/ml ethidium bromide for visualization of the DNAfragment. The fragment can then be purified from the agarose gel bydigestion with GELase™ (Epicentre Technologies) according to themanufacturer's instructions, ethanol precipitated, dried and resuspendedin 20 μL of water. Appropriate oligonucleotide adapters may be ligatedto the fragment using T4 DNA ligase (New England Biolabs, Beverly,Mass.). The fragment containing the ligated adapters can be purifiedfrom the excess adapters using low melting agarose as described above.The vector pBT430 is digested, dephosphorylated with alkalinephosphatase (NEB) and deproteinized with phenol/chloroform as describedabove. The prepared vector pBT430 and fragment can then be ligated at16° C. for 15 hours followed by transformation into DH5 electrocompetentcells (GIBCO BRL). Transformants can be selected on agar platescontaining LB media and 100 μg/mL ampicillin. Transformants containingthe gene encoding the instant polypeptides are then screened for thecorrect orientation with respect to the T7 promoter by restrictionenzyme analysis.

For high level expression, a plasmid clone with the cDNA insert in thecorrect orientation relative to the T7 promoter can be transformed intoE. coli strain BL21 (DE3) (Studier et al. (1986) J. Mol. Biol.189:113-130). Cultures are grown in LB medium containing ampicillin (100mg/L) at 25° C. At an optical density at 600 nm of approximately 1, IPTG(isopropylthio-β-galactoside, the inducer) can be added to a finalconcentration of 0.4 mM and incubation can be continued for 3 h at 25°.Cells are then harvested by centrifugation and re-suspended in 50 μL of50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenylmethylsulfonyl fluoride. A small amount of 1 mm glass beads can be addedand the mixture sonicated 3 times for about 5 seconds each time with amicroprobe sonicator. The mixture is centrifuged and the proteinconcentration of the supernatant determined. One jig of protein from thesoluble fraction of the culture can be separated by SDS-polyacrylamidegel electrophoresis. Gels can be observed for protein bands migrating atthe expected molecular weight.

Example 10 Expression of Chimeric Genes in Insect Cells

The cDNAs encoding the instant polypeptides may be introduced into thebaculovirus genome itself. For this purpose the cDNAs may be placedunder the control of the polyhedron promoter, the IE1 promoter, or anyother one of the baculovirus promoters. The DNA, together withappropriate leader sequences is then inserted into a baculovirustransfer vector using standard molecular cloning techniques. Followingtransformation of E. coil DH5α, isolated colonies are chosen and plasmidDNA is prepared and is analyzed by restriction enzyme analysis. Coloniescontaining the appropriate fragment are isolated, propagated, andplasmid DNA is prepared for cotransfection.

Spodoptera frugiperda cells (Sf-9) are propagated in ExCell® 401 media(JRH Biosciences, Lenexa, Kans.) supplemented with 3.0% fetal bovineserum. Lipofectin® (50 μL at 0.1 mg/mL, Gibco/BRL) is added to a 50 μLaliquot of the transfer vector containing the PDI gene (500 ng) andlinearized polyhedrin-negative AcNPV (2.5 μg, Baculogold® viral DNA,Pharmigen, San Diego, Calif.). Sf-9 cells (approximate 50% monolayer)are co-transfected with the viral DNA/transfer vector solution. Thesupernatant fluid from the co-transfection experiment is collected at 5days post-transfection and recombinant viruses are isolated employingstandard plaque purification protocols, wherein only polyhedrin-positiveplaques are selected (O'Reilly et al. (1992), Baculovirus ExpressionVectors: A Laboratory Manual, W. H. Freeman and Company, New York.).Sf-9 cells in 35 mM petri dishes (50% monolayer) are inoculated with 100μL of a serial dilution of the viral suspension, and supernatant fluidsare collected at 5 days post infection. In order to prepare largerquantities of virus for characterization, these supernatant fluids areused to inoculate larger tissue cultures for large scale propagation ofrecombinant viruses. Expression of the instant polypeptides encoded bythe recombinant baculovirus is confirmed by bioassay.

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 32 <210> SEQ ID NO 1 <211>LENGTH: 2149 <212> TYPE: DNA <213> ORGANISM: Argiope sp. <400> SEQUENCE:1 gctcgagttt tcctttctct tgcggcgttg gtgttttcag tgtcaagagt agcagttttc 60atataccggc tttctattta acacggagca atgtttatta aattactaat aactacgcta 120tgtattgcat ccactatcgc agatgaaatt aagaaagaaa atcatgtttt agttttgaca 180aatgataatt tcgaaggtgc aatcaaagat aaaaacgttc tggtcgaatt ctatgctcca 240tggtgtgggc attgtaaggc tcttgaaccc caatatgcca aagccgcaga gagtctagct 300gaagaaaaat ctgaactact cttggctaaa gttgatgcca cagttgaaac tgatcttgct 360gaacggtatg gagttcgtgg atacccaacc atcaaattct tccgtgaagg aaagatcttc 420gaatataatg gaggacgaac ttcagatgaa attatcagat ggcttaaaaa gaaaactgga 480cctcccgcag ttgatttatc atctgtagaa gatgccaaaa agtttgttga tagtaacgaa 540gttgctgttg ttgggttttt caaggatctt gaaagtgcag atgcaaagat tttcaagagt 600gttgcatcag aaatggatga cttcgtattc ggtattacag atgacgatgt tgtttattct 660gaacttaagg cctcaaagga tggagtcatt ctttttaaaa agtttgatga aggacgaaat 720gaatatgaag gtgaattgaa ggaagaggat ctgaaaaaat tcttgaaatc gaacagcttg 780ccattagtag ttgaattcag ccatgaaaca gcacaaaaga tctttggcgg agacattaaa 840gcacacaatc ttctgttcat cagtaaagaa tcttcagatt atgaaagccg tgtagatgtc 900ttccgtaagg tagcaaagga attcaaaggc aaggttttgt ttgttacaat caataccgat 960gatgaagatc atgaaaagat catggacttc tttggcttga agaaagaaga tactccaact 1020atgagattga tcaagcttga agaagaaatg gctaaattca agcctcctac cgaaggaaac 1080agcgaaagtg aaattagaga cttcgtaaat ggtgttttgg aaggaaagat aaagcaacat 1140ttactttctg aagacattcc tgaaggatgg gataaagaac ctgtaaaagt tcttgtagga 1200aagaattttg atgaagtggc ttttgacaaa tccaagaatg ttcttgttga attctatgct 1260ccatggtgtg gtcactgcaa gcagttggct cccatttatg atgagcttgg tgaaaagtac 1320aaagacagag atgatgttgt tattgccaaa atggacgcta cagcaaatga attggaacat 1380actaaaatca atagctatcc tactattaaa ttgtacaaaa aaggcacaaa tgaggttgtt 1440gagtacaatg gagaacgtac tttagaaggc attaataaat tcactgaaac tgatggagaa 1500tatggaaaag ctgctcccga tgaggaggaa gctgaagaag cagtcaggga agatgcgcag 1560gcacatcgtg atgaactata agcatcgcaa cttaataggt cattcatttc atactcctca 1620tacacgcatt gtgtaccaaa gtcaagcgca atcagttgtt aaactcattt ttccattcaa 1680gccaatggct ggtctccagg ggttaaaata aatcggacat tttgtgttgt ggttgtgctg 1740aacaaattgg atttaatgat actaataaaa aaaaaattgt tctgtaaaat atttttctta 1800tcagatttgt tagagattat atacaaagca ggcattttaa gtttcaatta tttcattatt 1860tttttatgct gatgttgtgt tcaaaaacgg tgtgaacaaa gatgttatgc tgtttttata 1920atttttttat ttaagtaaat gttaaatgat aacttttgaa gatttttcat ttattacgtt 1980atttaaatac atctgttcat acttttattt ttaatcaaat aattggcaat agaataaaat 2040attttttata taaaaaacat agttcatttt gaattaaatg ctcgtcttcc ttgtgtgtat 2100taatattaca ggtgtaataa aatacttgtt aaaaaaaaaa aaaaaaaaa 2149 <210> SEQ IDNO 2 <211> LENGTH: 496 <212> TYPE: PRT <213> ORGANISM: Argiope sp. <400>SEQUENCE: 2 Met Phe Ile Lys Leu Leu Ile Thr Thr Leu Cys Ile Ala Ser ThrIle 1 5 10 15 Ala Asp Glu Ile Lys Lys Glu Asn His Val Leu Val Leu ThrAsn Asp 20 25 30 Asn Phe Glu Gly Ala Ile Lys Asp Lys Asn Val Leu Val GluPhe Tyr 35 40 45 Ala Pro Trp Cys Gly His Cys Lys Ala Leu Glu Pro Gln TyrAla Lys 50 55 60 Ala Ala Glu Ser Leu Ala Glu Glu Lys Ser Glu Leu Leu LeuAla Lys 65 70 75 80 Val Asp Ala Thr Val Glu Thr Asp Leu Ala Glu Arg TyrGly Val Arg 85 90 95 Gly Tyr Pro Thr Ile Lys Phe Phe Arg Glu Gly Lys IlePhe Glu Tyr 100 105 110 Asn Gly Gly Arg Thr Ser Asp Glu Ile Ile Arg TrpLeu Lys Lys Lys 115 120 125 Thr Gly Pro Pro Ala Val Asp Leu Ser Ser ValGlu Asp Ala Lys Lys 130 135 140 Phe Val Asp Ser Asn Glu Val Ala Val ValGly Phe Phe Lys Asp Leu 145 150 155 160 Glu Ser Ala Asp Ala Lys Ile PheLys Ser Val Ala Ser Glu Met Asp 165 170 175 Asp Phe Val Phe Gly Ile ThrAsp Asp Asp Val Val Tyr Ser Glu Leu 180 185 190 Lys Ala Ser Lys Asp GlyVal Ile Leu Phe Lys Lys Phe Asp Glu Gly 195 200 205 Arg Asn Glu Tyr GluGly Glu Leu Lys Glu Glu Asp Leu Lys Lys Phe 210 215 220 Leu Lys Ser AsnSer Leu Pro Leu Val Val Glu Phe Ser His Glu Thr 225 230 235 240 Ala GlnLys Ile Phe Gly Gly Asp Ile Lys Ala His Asn Leu Leu Phe 245 250 255 IleSer Lys Glu Ser Ser Asp Tyr Glu Ser Arg Val Asp Val Phe Arg 260 265 270Lys Val Ala Lys Glu Phe Lys Gly Lys Val Leu Phe Val Thr Ile Asn 275 280285 Thr Asp Asp Glu Asp His Glu Lys Ile Met Asp Phe Phe Gly Leu Lys 290295 300 Lys Glu Asp Thr Pro Thr Met Arg Leu Ile Lys Leu Glu Glu Glu Met305 310 315 320 Ala Lys Phe Lys Pro Pro Thr Glu Gly Asn Ser Glu Ser GluIle Arg 325 330 335 Asp Phe Val Asn Gly Val Leu Glu Gly Lys Ile Lys GlnHis Leu Leu 340 345 350 Ser Glu Asp Ile Pro Glu Gly Trp Asp Lys Glu ProVal Lys Val Leu 355 360 365 Val Gly Lys Asn Phe Asp Glu Val Ala Phe AspLys Ser Lys Asn Val 370 375 380 Leu Val Glu Phe Tyr Ala Pro Trp Cys GlyHis Cys Lys Gln Leu Ala 385 390 395 400 Pro Ile Tyr Asp Glu Leu Gly GluLys Tyr Lys Asp Arg Asp Asp Val 405 410 415 Val Ile Ala Lys Met Asp AlaThr Ala Asn Glu Leu Glu His Thr Lys 420 425 430 Ile Asn Ser Tyr Pro ThrIle Lys Leu Tyr Lys Lys Gly Thr Asn Glu 435 440 445 Val Val Glu Tyr AsnGly Glu Arg Thr Leu Glu Gly Ile Asn Lys Phe 450 455 460 Thr Glu Thr AspGly Glu Tyr Gly Lys Ala Ala Pro Asp Glu Glu Glu 465 470 475 480 Ala GluGlu Ala Val Arg Glu Asp Ala Gln Ala His Arg Asp Glu Leu 485 490 495<210> SEQ ID NO 3 <211> LENGTH: 1843 <212> TYPE: DNA <213> ORGANISM:Scolopendra canidens DS <400> SEQUENCE: 3 gcacgagtat aaacgcctgcctctccgttt gcttctgctt gccacgtttg acttttcctc 60 taaccagcgg cattggtgcatgtttagtgt caggtgatag gcttttcatt taccggcttg 120 aacttaaatt tataaaatgtatataaagct tcttattctt agtttatgca tatatttatg 180 tatagctgat gaaattaaaaaagaaaagtc agttttagtg ttgaccaaag acaattttga 240 aggagcaatc aaagataaaagtgtactcgt cgagttttat gctccttggt gtggtcattg 300 taaggcccta gaacctgaatatgctaaagc cgctcagatc ttggaagagg aaaagtccga 360 cttactgttg gctaaagttgatgctacagc ggaaactgat ttagctgaac agcatggcgt 420 tcgtgggtat ccaactattaagtttttccg agagggtaaa gtgatagaat actctggtgg 480 acgaactgct gatgacattatacgctggct gaaaaagaag actgggcctc cagcaactga 540 tttaactaca gtagaggcaacaaaatcttt tattgatggt ggtgaggtag tagttgtagg 600 cttctttaaa gaccaaaattcagatcaggc aaagattttc aagaatgttg ctgcagaaat 660 ggatgacttt gttttcggcattacttcaac tgatgaggtg tacaatgaat taaaggctac 720 acaagatggc gttgttttgtttaaaaagtt tgatgaagga aggaatgttt atgaaggtga 780 attatcagaa gaaaaattgaaaaaattctt gaaatctaat agcctgcctc tagttgttga 840 gtttacccat gaatcagctcagaaaatctt tgggggtgac attaaagccc ataatttact 900 cttcatcagc aaaggaacctcggattatga aagcaaaatt gaagccttcc gtaaagtcgc 960 taaagaattc aaaggcaaagttctttttgt tactatcgat actgatgatg aagatcatga 1020 gagaatcatg gaattctttggtcttaaaaa agaagaggcg cctaccatga gactgatcaa 1080 actagaagat gaaatgactaaattcaaacc tactactaca ggaattgagg aaagtgatat 1140 tagaggattt gttactggtgttttagaagg aaagataaag caacacttgc tttctgaaga 1200 tgttcctgaa ggatgggataaagagcctgt gaaaattcta gttggaaaaa attttgatga 1260 agtagtattt gataaaacaaagaatgttct agttgaattt tatgctccat ggtgtggcca 1320 ttgtaaacag ctagctcctatttatgatga attgggtgag aaatacaagg atcaagctga 1380 tattgtaatt gctaagatggatgctactgc aaatgaattg gagcatacca agattaacag 1440 ttttcccaca atcaagttgtacaaaaaaga tacaaatgaa gtggttgatt tcaatggaga 1500 acgcacactt gaaggaattagcaggtttat tgatactggt ggtgttgatg gggcagctgt 1560 taaagaagag gaggaagatgaggaagaaga aaaagatgac gaacaggcca agcgtgatga 1620 gctttaaagg cattatcacaagacttaagt catcatctcc atttttctca tacaacattg 1680 tgtaccaaag ttgagtgcgatcagttcttg taaccattaa tttcattcat gccaatggct 1740 ggattccagg ggttgtaacaaattaaggac ctttttcttg ttcagtgaat gtgccgaagc 1800 agttctaata aaatatttgttttttaaaaa aaaaaaaaaa aaa 1843 <210> SEQ ID NO 4 <211> LENGTH: 496 <212>TYPE: PRT <213> ORGANISM: Scolopendra canidens DS <400> SEQUENCE: 4 MetTyr Ile Lys Leu Leu Ile Leu Ser Leu Cys Ile Tyr Leu Cys Ile 1 5 10 15Ala Asp Glu Ile Lys Lys Glu Lys Ser Val Leu Val Leu Thr Lys Asp 20 25 30Asn Phe Glu Gly Ala Ile Lys Asp Lys Ser Val Leu Val Glu Phe Tyr 35 40 45Ala Pro Trp Cys Gly His Cys Lys Ala Leu Glu Pro Glu Tyr Ala Lys 50 55 60Ala Ala Gln Ile Leu Glu Glu Glu Lys Ser Asp Leu Leu Leu Ala Lys 65 70 7580 Val Asp Ala Thr Ala Glu Thr Asp Leu Ala Glu Gln His Gly Val Arg 85 9095 Gly Tyr Pro Thr Ile Lys Phe Phe Arg Glu Gly Lys Val Ile Glu Tyr 100105 110 Ser Gly Gly Arg Thr Ala Asp Asp Ile Ile Arg Trp Leu Lys Lys Lys115 120 125 Thr Gly Pro Pro Ala Thr Asp Leu Thr Thr Val Glu Ala Thr LysSer 130 135 140 Phe Ile Asp Gly Gly Glu Val Val Val Val Gly Phe Phe LysAsp Gln 145 150 155 160 Asn Ser Asp Gln Ala Lys Ile Phe Lys Asn Val AlaAla Glu Met Asp 165 170 175 Asp Phe Val Phe Gly Ile Thr Ser Thr Asp GluVal Tyr Asn Glu Leu 180 185 190 Lys Ala Thr Gln Asp Gly Val Val Leu PheLys Lys Phe Asp Glu Gly 195 200 205 Arg Asn Val Tyr Glu Gly Glu Leu SerGlu Glu Lys Leu Lys Lys Phe 210 215 220 Leu Lys Ser Asn Ser Leu Pro LeuVal Val Glu Phe Thr His Glu Ser 225 230 235 240 Ala Gln Lys Ile Phe GlyGly Asp Ile Lys Ala His Asn Leu Leu Phe 245 250 255 Ile Ser Lys Gly ThrSer Asp Tyr Glu Ser Lys Ile Glu Ala Phe Arg 260 265 270 Lys Val Ala LysGlu Phe Lys Gly Lys Val Leu Phe Val Thr Ile Asp 275 280 285 Thr Asp AspGlu Asp His Glu Arg Ile Met Glu Phe Phe Gly Leu Lys 290 295 300 Lys GluGlu Ala Pro Thr Met Arg Leu Ile Lys Leu Glu Asp Glu Met 305 310 315 320Thr Lys Phe Lys Pro Thr Thr Thr Gly Ile Glu Glu Ser Asp Ile Arg 325 330335 Gly Phe Val Thr Gly Val Leu Glu Gly Lys Ile Lys Gln His Leu Leu 340345 350 Ser Glu Asp Val Pro Glu Gly Trp Asp Lys Glu Pro Val Lys Ile Leu355 360 365 Val Gly Lys Asn Phe Asp Glu Val Val Phe Asp Lys Thr Lys AsnVal 370 375 380 Leu Val Glu Phe Tyr Ala Pro Trp Cys Gly His Cys Lys GlnLeu Ala 385 390 395 400 Pro Ile Tyr Asp Glu Leu Gly Glu Lys Tyr Lys AspGln Ala Asp Ile 405 410 415 Val Ile Ala Lys Met Asp Ala Thr Ala Asn GluLeu Glu His Thr Lys 420 425 430 Ile Asn Ser Phe Pro Thr Ile Lys Leu TyrLys Lys Asp Thr Asn Glu 435 440 445 Val Val Asp Phe Asn Gly Glu Arg ThrLeu Glu Gly Ile Ser Arg Phe 450 455 460 Ile Asp Thr Gly Gly Val Asp GlyAla Ala Val Lys Glu Glu Glu Glu 465 470 475 480 Asp Glu Glu Glu Glu LysAsp Asp Glu Gln Ala Lys Arg Asp Glu Leu 485 490 495 <210> SEQ ID NO 5<211> LENGTH: 497 <212> TYPE: DNA <213> ORGANISM: Heliothus virescens]<220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (452) <400>SEQUENCE: 5 gtttcgtctt cggagcaggc ttgtgaagtg ccgtattttc caacatgagagtgatcctat 60 ttacggcgtt agcccttctg ggcaccgctt tggccgatga agtacccactgaagaaaacg 120 tactcgtttt aagcaaatct aacttcgaag gagtcatttc agcaaacaacttcatattag 180 tggaattcta tgcgccatgg tgcggtcact gcaagtccct cgctccggagtacgccaagg 240 ccgccaccaa gctggccgag gaggagtctc ccatcaaact ggccaaggttgatgccacgc 300 aagagcaaga gctcgctgag agctacggcg tcaggggata cccgaccctcaagttcttca 360 gaaacggcag ccctattgat tacactggtg gtcgccaagc tgacgacattgtctcctggc 420 tgaagaagaa gactggtcct cccgcccttg anggtgcctc ctgctgagcaagccaaggaa 480 ctcaattgcc gccaaca 497 <210> SEQ ID NO 6 <211> LENGTH:133 <212> TYPE: PRT <213> ORGANISM: Heliothus virescens] <400> SEQUENCE:6 Met Arg Val Ile Leu Phe Thr Ala Leu Ala Leu Leu Gly Thr Ala Leu 1 5 1015 Ala Asp Glu Val Pro Thr Glu Glu Asn Val Leu Val Leu Ser Lys Ser 20 2530 Asn Phe Glu Gly Val Ile Ser Ala Asn Asn Phe Ile Leu Val Glu Phe 35 4045 Tyr Ala Pro Trp Cys Gly His Cys Lys Ser Leu Ala Pro Glu Tyr Ala 50 5560 Lys Ala Ala Thr Lys Leu Ala Glu Glu Glu Ser Pro Ile Lys Leu Ala 65 7075 80 Lys Val Asp Ala Thr Gln Glu Gln Glu Leu Ala Glu Ser Tyr Gly Val 8590 95 Arg Gly Tyr Pro Thr Leu Lys Phe Phe Arg Asn Gly Ser Pro Ile Asp100 105 110 Tyr Thr Gly Gly Arg Gln Ala Asp Asp Ile Val Ser Trp Leu LysLys 115 120 125 Lys Thr Gly Pro Pro 130 <210> SEQ ID NO 7 <211> LENGTH:1859 <212> TYPE: DNA <213> ORGANISM: Vaejovis carolinianus <400>SEQUENCE: 7 gcacgagttt tagtagaaaa atgttgggtg cggttacttt atctacaattttactagttg 60 ttatagcagc agcagatgaa atcaagaaag acggcgaagt tctagttttaaacaaagaca 120 atttccaaaa agctattcaa gaaaacaaac atatcttggt tgaattttatgcaccatggt 180 gtggccactg taaagcactt gagccagaat atgccaaagc agccaagcagcttaaagaag 240 aacagtctga tattgctctt ggtaagattg atgccacagc agagagtgagttagcggaag 300 agtatgatgt ccgaggatat cctacaataa aattcatcag ggatggcaaaccttcagagt 360 acaaaggtgg tcgaacagcg gaagatattg ttcgctggct gaagaagaaagttggtcctc 420 cagctgaaaa tcttgacaca gttgacagtg tcaaaacatt ccaatcaagtgcagaagttg 480 tattagttgg tttctttaaa gatcaatcat cagataaagc taaggtgttcttggaggtag 540 ctttagaatc tgatgactat gcttttggaa tcacatcaca ggatgatgtgtttaaggcat 600 ataatgtgga aaaagatgga attgttttgt ttaaacagtt tgatgaaggaagaaatgact 660 ttgaaggaga aataacagca gatgctttaa aagagtttat taatgctaacagcttgccat 720 tggttgttga gttcaatcaa gatactgcac agaaagtctt tggtggtgaaatcaaagctc 780 acaatttact ttttgtcagc aaacagcaga gtgaagagta tgagaaattacttgaagtct 840 tccgtaaagt tgccaaagat tttaagaata aggtattgtt tgttacaattgacatagatg 900 aagaggacca tgaaagaata atggaatttt ttggaatgaa gaaagaagatgcaccagata 960 tgcgtttgat cagattagaa gaggagatga caaagtttaa accaccatctcctggactct 1020 cagaagaaaa tatacgctct tttgttcagg gagttttaga tggaaaaatcaagaggcact 1080 tgctgtctga aagtgttcca gatgactggg acaagggagc agttaaggttctggttggac 1140 aaaacttcga tgatgttgca tttgacaaat caaaagatgt gcttgttgagttttatgcac 1200 cttggtgtgg acattgcaaa cagcttgcac ccatctatga agaacttggagagaagtaca 1260 aagatcagtc atctattgtt attgcaaaaa tggatgccac taccaatgaactggaccatg 1320 taaaaatcca tagttttccc acaattaaat tattcaaaaa agataccaatgaggtcatag 1380 acttcaatgg tgaacgcaca cttgaaggac tgacaaaatt tattgattcaggaggagtgg 1440 atggcgcttc accaaaggaa gaggagattg acgaagagga ggaaaaggatgatgatgaaa 1500 agaaaaggga cgaactgtga tttttcactg aaaactaatt tacacatgttgcttcattgc 1560 agcagatttt gtccattcct gaattttcct gtgtacagaa aaggttgtgttgaacaatag 1620 gttttggcaa catttaacgt aacaaacaag gcttgctaag tattgccatttatcaaatgt 1680 taaaatgcca aagcatattt gtaggaagtc tttttaattt aaaatatttttcccctaact 1740 ttttattttt tatcttttat taaaatgaat gacttttaaa tcatcggtggtgcaaatgct 1800 ggtcgtggaa attatttgga atgctaaaac aaagcaaaac aaattgcgatggtcgtact 1859 <210> SEQ ID NO 8 <211> LENGTH: 499 <212> TYPE: PRT <213>ORGANISM: Vaejovis carolinianus <400> SEQUENCE: 8 Met Leu Gly Ala ValThr Leu Ser Thr Ile Leu Leu Val Val Ile Ala 1 5 10 15 Ala Ala Asp GluIle Lys Lys Asp Gly Glu Val Leu Val Leu Asn Lys 20 25 30 Asp Asn Phe GlnLys Ala Ile Gln Glu Asn Lys His Ile Leu Val Glu 35 40 45 Phe Tyr Ala ProTrp Cys Gly His Cys Lys Ala Leu Glu Pro Glu Tyr 50 55 60 Ala Lys Ala AlaLys Gln Leu Lys Glu Glu Gln Ser Asp Ile Ala Leu 65 70 75 80 Gly Lys IleAsp Ala Thr Ala Glu Ser Glu Leu Ala Glu Glu Tyr Asp 85 90 95 Val Arg GlyTyr Pro Thr Ile Lys Phe Ile Arg Asp Gly Lys Pro Ser 100 105 110 Glu TyrLys Gly Gly Arg Thr Ala Glu Asp Ile Val Arg Trp Leu Lys 115 120 125 LysLys Val Gly Pro Pro Ala Glu Asn Leu Asp Thr Val Asp Ser Val 130 135 140Lys Thr Phe Gln Ser Ser Ala Glu Val Val Leu Val Gly Phe Phe Lys 145 150155 160 Asp Gln Ser Ser Asp Lys Ala Lys Val Phe Leu Glu Val Ala Leu Glu165 170 175 Ser Asp Asp Tyr Ala Phe Gly Ile Thr Ser Gln Asp Asp Val PheLys 180 185 190 Ala Tyr Asn Val Glu Lys Asp Gly Ile Val Leu Phe Lys GlnPhe Asp 195 200 205 Glu Gly Arg Asn Asp Phe Glu Gly Glu Ile Thr Ala AspAla Leu Lys 210 215 220 Glu Phe Ile Asn Ala Asn Ser Leu Pro Leu Val ValGlu Phe Asn Gln 225 230 235 240 Asp Thr Ala Gln Lys Val Phe Gly Gly GluIle Lys Ala His Asn Leu 245 250 255 Leu Phe Val Ser Lys Gln Gln Ser GluGlu Tyr Glu Lys Leu Leu Glu 260 265 270 Val Phe Arg Lys Val Ala Lys AspPhe Lys Asn Lys Val Leu Phe Val 275 280 285 Thr Ile Asp Ile Asp Glu GluAsp His Glu Arg Ile Met Glu Phe Phe 290 295 300 Gly Met Lys Lys Glu AspAla Pro Asp Met Arg Leu Ile Arg Leu Glu 305 310 315 320 Glu Glu Met ThrLys Phe Lys Pro Pro Ser Pro Gly Leu Ser Glu Glu 325 330 335 Asn Ile ArgSer Phe Val Gln Gly Val Leu Asp Gly Lys Ile Lys Arg 340 345 350 His LeuLeu Ser Glu Ser Val Pro Asp Asp Trp Asp Lys Gly Ala Val 355 360 365 LysVal Leu Val Gly Gln Asn Phe Asp Asp Val Ala Phe Asp Lys Ser 370 375 380Lys Asp Val Leu Val Glu Phe Tyr Ala Pro Trp Cys Gly His Cys Lys 385 390395 400 Gln Leu Ala Pro Ile Tyr Glu Glu Leu Gly Glu Lys Tyr Lys Asp Gln405 410 415 Ser Ser Ile Val Ile Ala Lys Met Asp Ala Thr Thr Asn Glu LeuAsp 420 425 430 His Val Lys Ile His Ser Phe Pro Thr Ile Lys Leu Phe LysLys Asp 435 440 445 Thr Asn Glu Val Ile Asp Phe Asn Gly Glu Arg Thr LeuGlu Gly Leu 450 455 460 Thr Lys Phe Ile Asp Ser Gly Gly Val Asp Gly AlaSer Pro Lys Glu 465 470 475 480 Glu Glu Ile Asp Glu Glu Glu Glu Lys AspAsp Asp Glu Lys Lys Arg 485 490 495 Asp Glu Leu <210> SEQ ID NO 9 <211>LENGTH: 2170 <212> TYPE: DNA <213> ORGANISM: Argiope sp. <400> SEQUENCE:9 gcacgagaaa aagtataagc atcagtgaac cgctgaagtc tattgatttt taccaaaaga 60tttacaatga agctgttatt tctgtcatca atacttagtt gtacattatc tgtaatactt 120tgtagtgatg tcctagattt ttccggtgcc gatttcgaag atcgaatagc agaacatgat 180gcaattttag ttgaattctt cgctccttgg tgtggacatt gtaagagatt agcacctgaa 240tatgacaaag catctacgat attaaaaaaa gctgacccac ctataccact agcaaaggtt 300gactgcacat cagataatgg taaagataca tgttcaaaat atggtgtaag tggttatcct 360actttaaaga tatttagagg tggagaattt tcttcagaat ataatggtcc tcgtgatgca 420gatggtattg ttaagtatat gaaggctcaa gttggtccaa gttcaaaaga acttcaatct 480ttggaagatg cagagaaaat tttaaaagat gatattgttg tgattggtta ctttgctgat 540tcgtcaaata aattaaagga agaattctta aaagcagctg ataaactcag agaatctgta 600tcttttgcac atacatctag taaagacatt ttagacaaat atgggtattc tgatgaaatt 660gttttatacc gtcccaagat cttctggagt aaatttgaac cccaagaaat aaaatataca 720ggtgatgcag ataaaggaga gatttctcaa tttattaaag acaactacca tggtcttgtt 780ggtcatagaa cacatgacaa tcacgatgac tttaaggctc ctcttattgt tgtatattat 840gatgtagact atgtaaagaa tgtaaaaggc acaaactact ggcgtaacag agtaatgaag 900gttgcacaga actacaaggg acaagtcaac tttgctatta gtaacaaaga caaattttct 960gctgaagttg aagattttgg tttaaaagct acaggagata aacctgtagt tgctgccaga 1020aatgataaaa accagaaatt taatatgaag gaagaattca gtgttgaaaa ttttgaaact 1080tttgtgaaaa aatttttgga tggcagttta gaaccacact tgaaatctga acctgttcct 1140gaaaagaacg acgggcctgt taaagtagct gttgcccaaa actttgaaga acttgtaatg 1200gaaaatgaca aagatgtatt gatcgaattt tatgccccat ggtgtggaca ttgtaagaaa 1260ttggctccta cttatgaaga acttggtcaa aagttggaag gtgaagatgt agagattgtg 1320aaaatggatg ctacagctaa tgatgttcca cctacatttg aggttcatgg attcccaaca 1380ttatattggg tacctaaaac acacaagtct agtccgaaga aatatgaagg tggccgagaa 1440atcaaggact ttatcaatta cattgccaag catgctacaa atgaattgaa acaatatgac 1500cgatctggca aaaagaagtc caaggaagag ctataagtta atatcactct tttaggaact 1560atttttcaat gtgcatagtt tgtgttaagc atcaatcatg ttaatcttgt tacatattaa 1620ttgaacaaat gtggttaggc atgtactgat catctgttca tacgttccaa attgactctg 1680tgggacctaa agtgtctttt tgtaataaaa cgttttttta tacttctgct gttttttttc 1740cccttacgag tggaattttt ttacttttta cttttattct ttattaatgg gtattgagtc 1800tgaaattatt tatttagtct tttattaatc ttttccttgt tattttaaga ttgaatatct 1860actaaacatt ttctttaaaa aaatctatgc aatttgtaat atgccatcat gaatttcatg 1920gaagttcttg tatattcatt agatcaatca aaatatattt gcagtgcaga tatttaaagc 1980tgaacaattt cttcgtagag taatacacat gtatttaaaa tgatctgcaa aagccttaac 2040tttgaatgtt acttgtacat ttgttgcaaa atagcatttg tcgtatttta tgtaattttt 2100ctatttaaat cgattgaaat aaaggtagtt cttttaaaaa aaaaaaaaaa aaaaaaaaaa 2160aaaaaaaaaa 2170 <210> SEQ ID NO 10 <211> LENGTH: 489 <212> TYPE: PRT<213> ORGANISM: Argiope sp. <400> SEQUENCE: 10 Met Lys Leu Leu Phe LeuSer Ser Ile Leu Ser Cys Thr Leu Ser Val 1 5 10 15 Ile Leu Cys Ser AspVal Leu Asp Phe Ser Gly Ala Asp Phe Glu Asp 20 25 30 Arg Ile Ala Glu HisAsp Ala Ile Leu Val Glu Phe Phe Ala Pro Trp 35 40 45 Cys Gly His Cys LysArg Leu Ala Pro Glu Tyr Asp Lys Ala Ser Thr 50 55 60 Ile Leu Lys Lys AlaAsp Pro Pro Ile Pro Leu Ala Lys Val Asp Cys 65 70 75 80 Thr Ser Asp AsnGly Lys Asp Thr Cys Ser Lys Tyr Gly Val Ser Gly 85 90 95 Tyr Pro Thr LeuLys Ile Phe Arg Gly Gly Glu Phe Ser Ser Glu Tyr 100 105 110 Asn Gly ProArg Asp Ala Asp Gly Ile Val Lys Tyr Met Lys Ala Gln 115 120 125 Val GlyPro Ser Ser Lys Glu Leu Gln Ser Leu Glu Asp Ala Glu Lys 130 135 140 IleLeu Lys Asp Asp Ile Val Val Ile Gly Tyr Phe Ala Asp Ser Ser 145 150 155160 Asn Lys Leu Lys Glu Glu Phe Leu Lys Ala Ala Asp Lys Leu Arg Glu 165170 175 Ser Val Ser Phe Ala His Thr Ser Ser Lys Asp Ile Leu Asp Lys Tyr180 185 190 Gly Tyr Ser Asp Glu Ile Val Leu Tyr Arg Pro Lys Ile Phe TrpSer 195 200 205 Lys Phe Glu Pro Gln Glu Ile Lys Tyr Thr Gly Asp Ala AspLys Gly 210 215 220 Glu Ile Ser Gln Phe Ile Lys Asp Asn Tyr His Gly LeuVal Gly His 225 230 235 240 Arg Thr His Asp Asn His Asp Asp Phe Lys AlaPro Leu Ile Val Val 245 250 255 Tyr Tyr Asp Val Asp Tyr Val Lys Asn ValLys Gly Thr Asn Tyr Trp 260 265 270 Arg Asn Arg Val Met Lys Val Ala GlnAsn Tyr Lys Gly Gln Val Asn 275 280 285 Phe Ala Ile Ser Asn Lys Asp LysPhe Ser Ala Glu Val Glu Asp Phe 290 295 300 Gly Leu Lys Ala Thr Gly AspLys Pro Val Val Ala Ala Arg Asn Asp 305 310 315 320 Lys Asn Gln Lys PheAsn Met Lys Glu Glu Phe Ser Val Glu Asn Phe 325 330 335 Glu Thr Phe ValLys Lys Phe Leu Asp Gly Ser Leu Glu Pro His Leu 340 345 350 Lys Ser GluPro Val Pro Glu Lys Asn Asp Gly Pro Val Lys Val Ala 355 360 365 Val AlaGln Asn Phe Glu Glu Leu Val Met Glu Asn Asp Lys Asp Val 370 375 380 LeuIle Glu Phe Tyr Ala Pro Trp Cys Gly His Cys Lys Lys Leu Ala 385 390 395400 Pro Thr Tyr Glu Glu Leu Gly Gln Lys Leu Glu Gly Glu Asp Val Glu 405410 415 Ile Val Lys Met Asp Ala Thr Ala Asn Asp Val Pro Pro Thr Phe Glu420 425 430 Val His Gly Phe Pro Thr Leu Tyr Trp Val Pro Lys Thr His LysSer 435 440 445 Ser Pro Lys Lys Tyr Glu Gly Gly Arg Glu Ile Lys Asp PheIle Asn 450 455 460 Tyr Ile Ala Lys His Ala Thr Asn Glu Leu Lys Gln TyrAsp Arg Ser 465 470 475 480 Gly Lys Lys Lys Ser Lys Glu Glu Leu 485<210> SEQ ID NO 11 <211> LENGTH: 1584 <212> TYPE: DNA <213> ORGANISM:Hottentotta judiaca <400> SEQUENCE: 11 gcacgagact tggtccaaaa tatgaagaggcagcaacaat tttgaagaaa aatgatccac 60 ctgttccttt agctaaggtg gattgtacatctgatgctgg aaaagaaact tgttcaaaat 120 atggtgtcag tggctatcct actctcaagattttccgaaa tggtgaattt tcttcagagt 180 acagtggtgg gcgagaaaca gatgctattgtaaaatatat gaaatctcaa gttggaccaa 240 gctctgtaga aattaaaaca cctgctgatgctaaaaagtt attaagcaga atcgaagtag 300 ttataattgg attttttaag gatgaaaaaagtcagttgaa agaagaattt ttaaaagttg 360 cagataaaat gagagagacc acttcctttggacacacatc taatcaagaa gttcttgatt 420 tatatggata taaagatcaa atagtgctcttccgtcctca gcatcttcaa agcaaatttg 480 aagaaaaaga attaaaatat gaaggtggtgctgaaaaatc aaaaatagaa gatttcatta 540 gagaaaatta tcatggattg gttggtcatcgaactagtga taatttccaa gatttcaaga 600 atcctcttgt tgtagcatat tatgatgtagactatgtaaa aaatactaaa ggtacaaatt 660 attggcgtaa tcgtatcatg aaagtagcacaacatttcaa agacaaattg aactttgctg 720 tatctaatat aaatcaattt tctgctgaaattgaagaatt tggcctaact gttaaaggtg 780 ataagcctgc aattgcagta cgtaatgaaaagcaacagaa attccgaatg actgatgaat 840 ttagcatgga tgcatttgag aaatttcttaaggacttttt agatggaaag ttagaagcac 900 acgtaaaatc tgaaccaatt ccagaaaataatgatggacc agttaaggtt gcagttgcat 960 caaattttga tgatattgta acaaataatgataaagacat cttgctagaa ttttatgctc 1020 catggtgtgg acattgtaaa aaacttgctccaacatatga agaattaggc acagagatga 1080 aacaggaaga tgttgaaata gttaaaatggatgcaacagc aaatgatgtt cctccacctt 1140 atgaagttca tgggtttcca acactttattgggttccaaa aaatagcaaa aataatccca 1200 aaaaatatga tggtggtcga gaattggatgatttaataaa atatatatct aaacacgcca 1260 caaatgaatt gaaaggatgg gacaggaagggcactaaaaa gtctgagaag acagagctct 1320 gagctgttct gtctaaattt ttcacagttgttagtgataa attcattttt gtgatgtcca 1380 aaattcattt gatttttagt ttacttgggacttcatccac tgactatcat tcctgcagtt 1440 tgtcattaat agcaactggg ggggttggattaagtgtaat gtttaatgaa tatttgtgaa 1500 ttttgaaggt aaatgttaaa taaatgtcaattttgtttac aaaaaaaaaa aaaaaaaaaa 1560 aaaaaaaaaa aaaaaaaaaa aaaa 1584<210> SEQ ID NO 12 <211> LENGTH: 439 <212> TYPE: PRT <213> ORGANISM:Hottentotta judiaca <400> SEQUENCE: 12 Thr Arg Leu Gly Pro Lys Tyr GluGlu Ala Ala Thr Ile Leu Lys Lys 1 5 10 15 Asn Asp Pro Pro Val Pro LeuAla Lys Val Asp Cys Thr Ser Asp Ala 20 25 30 Gly Lys Glu Thr Cys Ser LysTyr Gly Val Ser Gly Tyr Pro Thr Leu 35 40 45 Lys Ile Phe Arg Asn Gly GluPhe Ser Ser Glu Tyr Ser Gly Gly Arg 50 55 60 Glu Thr Asp Ala Ile Val LysTyr Met Lys Ser Gln Val Gly Pro Ser 65 70 75 80 Ser Val Glu Ile Lys ThrPro Ala Asp Ala Lys Lys Leu Leu Ser Arg 85 90 95 Ile Glu Val Val Ile IleGly Phe Phe Lys Asp Glu Lys Ser Gln Leu 100 105 110 Lys Glu Glu Phe LeuLys Val Ala Asp Lys Met Arg Glu Thr Thr Ser 115 120 125 Phe Gly His ThrSer Asn Gln Glu Val Leu Asp Leu Tyr Gly Tyr Lys 130 135 140 Asp Gln IleVal Leu Phe Arg Pro Gln His Leu Gln Ser Lys Phe Glu 145 150 155 160 GluLys Glu Leu Lys Tyr Glu Gly Gly Ala Glu Lys Ser Lys Ile Glu 165 170 175Asp Phe Ile Arg Glu Asn Tyr His Gly Leu Val Gly His Arg Thr Ser 180 185190 Asp Asn Phe Gln Asp Phe Lys Asn Pro Leu Val Val Ala Tyr Tyr Asp 195200 205 Val Asp Tyr Val Lys Asn Thr Lys Gly Thr Asn Tyr Trp Arg Asn Arg210 215 220 Ile Met Lys Val Ala Gln His Phe Lys Asp Lys Leu Asn Phe AlaVal 225 230 235 240 Ser Asn Ile Asn Gln Phe Ser Ala Glu Ile Glu Glu PheGly Leu Thr 245 250 255 Val Lys Gly Asp Lys Pro Ala Ile Ala Val Arg AsnGlu Lys Gln Gln 260 265 270 Lys Phe Arg Met Thr Asp Glu Phe Ser Met AspAla Phe Glu Lys Phe 275 280 285 Leu Lys Asp Phe Leu Asp Gly Lys Leu GluAla His Val Lys Ser Glu 290 295 300 Pro Ile Pro Glu Asn Asn Asp Gly ProVal Lys Val Ala Val Ala Ser 305 310 315 320 Asn Phe Asp Asp Ile Val ThrAsn Asn Asp Lys Asp Ile Leu Leu Glu 325 330 335 Phe Tyr Ala Pro Trp CysGly His Cys Lys Lys Leu Ala Pro Thr Tyr 340 345 350 Glu Glu Leu Gly ThrGlu Met Lys Gln Glu Asp Val Glu Ile Val Lys 355 360 365 Met Asp Ala ThrAla Asn Asp Val Pro Pro Pro Tyr Glu Val His Gly 370 375 380 Phe Pro ThrLeu Tyr Trp Val Pro Lys Asn Ser Lys Asn Asn Pro Lys 385 390 395 400 LysTyr Asp Gly Gly Arg Glu Leu Asp Asp Leu Ile Lys Tyr Ile Ser 405 410 415Lys His Ala Thr Asn Glu Leu Lys Gly Trp Asp Arg Lys Gly Thr Lys 420 425430 Lys Ser Glu Lys Thr Glu Leu 435 <210> SEQ ID NO 13 <211> LENGTH:1759 <212> TYPE: DNA <213> ORGANISM: Scolopendra canidens DS <400>SEQUENCE: 13 gcacgaggtg aattgccatt tcaccgggtg gagatagaac ggaatttatattttttttaa 60 ttgaaaatgt ggaaaatcgt tgctttttcc tgcttttttg tagcgactattgctagtgat 120 gtcttagaat ttaccgattc tgattttgac gaaaggatca aagaacatgatacttattta 180 gtcgagtttt atgcaccatg gtgtgggcat tgtaaacgct tagctccagaatatgaaaaa 240 gcagctacaa ttctaaaaga taatgatcca cccattcctc ttgttaaggttgattgtatc 300 gaatctggaa aagaaacttg tgggaaattt ggagtttccg gttacccaactctaaaaatt 360 tttagaaatg gagacttttc acaagaatac aatggaccaa gagaagcaaatggaattgta 420 aaatatatgg ctgcccaagt tgggccaagt tcgaaagaat tccagaatgtgaaagaagta 480 cagcaattcc ttgaaaaaga agaagttgca attattggat tttttgaatctgaagatgct 540 aaactcaaag caacattcca aaaagtagca gataaattga gagaaactgcaagatttggt 600 cattcttaca attcattggt gttgaaggaa tatggttata caaatcaagttgtattattt 660 cggcctaaac atcttcaaag taagtttgaa gattctcaag tggtatatgatggagacaaa 720 agtgataaac aggagcttga agaatttgtt aacaaaaatt accatggtttagtgggtcat 780 aggacaacag acaatactaa tcaattctct cctccattga tagtatcatattataaagtt 840 gattatgtta agaacaccaa aggtaccaat tattggcgta atcgtatcatgaaagttgca 900 tcagaattca aagggcgctt gaattttgcc atttcaaata aagatgaatttacacatgaa 960 ttaagcgaat atggttttaa ctatgtggct ggagataaac cagttgttgctgctcgaaat 1020 gcaaaatctg agaaatttgt aatggaaggc gaattttcaa taccaagctttgaaaagttc 1080 attaaggact ttttagatga aaaactaaaa ccatacttaa agtctgagccaattccagag 1140 aaaaatgaag agcctgttaa ggttgctgtt gctcaaaatt ttgaagagttagtcaccaag 1200 agtgataaag atgtattgat tgaattctat gctccatggt gtggacattgtaaaaaatta 1260 gcacctgttt atgatgaatt aggcaaagcc ttggaaggag aaacaacagtagaaatagtt 1320 aaaatggatg ccacagcaaa tgatgtgcct tcaccttatg aagtgcatggtttccctact 1380 ttatattggg ctccacgtga taagaaagat aagcctgtcc gatacgatggtggacgagaa 1440 ttggatgatt tcattaaata tattgctaag cattccacag atgaacttaaaacttacaac 1500 agaaatggga aaaagaaaaa agttgaattg taaagtagca atttagaatttaaaatattt 1560 gttcagtaaa agcacaattt tttattttta agggaataaa aatgtaaaaatcaattatga 1620 taatttaaaa tttagagtgc tttatgttgg tggtacgtat tgtctttatttctttgtaag 1680 ataaaatgtt gaaaattaat tcgaaataaa atttttttaa taaattagtttttacaaaaa 1740 gaaaaaaaaa aaaaaaaaa 1759 <210> SEQ ID NO 14 <211>LENGTH: 488 <212> TYPE: PRT <213> ORGANISM: Scolopendra canidens DS<400> SEQUENCE: 14 Met Trp Lys Ile Val Ala Phe Ser Cys Phe Phe Val AlaThr Ile Ala 1 5 10 15 Ser Asp Val Leu Glu Phe Thr Asp Ser Asp Phe AspGlu Arg Ile Lys 20 25 30 Glu His Asp Thr Tyr Leu Val Glu Phe Tyr Ala ProTrp Cys Gly His 35 40 45 Cys Lys Arg Leu Ala Pro Glu Tyr Glu Lys Ala AlaThr Ile Leu Lys 50 55 60 Asp Asn Asp Pro Pro Ile Pro Leu Val Lys Val AspCys Ile Glu Ser 65 70 75 80 Gly Lys Glu Thr Cys Gly Lys Phe Gly Val SerGly Tyr Pro Thr Leu 85 90 95 Lys Ile Phe Arg Asn Gly Asp Phe Ser Gln GluTyr Asn Gly Pro Arg 100 105 110 Glu Ala Asn Gly Ile Val Lys Tyr Met AlaAla Gln Val Gly Pro Ser 115 120 125 Ser Lys Glu Phe Gln Asn Val Lys GluVal Gln Gln Phe Leu Glu Lys 130 135 140 Glu Glu Val Ala Ile Ile Gly PhePhe Glu Ser Glu Asp Ala Lys Leu 145 150 155 160 Lys Ala Thr Phe Gln LysVal Ala Asp Lys Leu Arg Glu Thr Ala Arg 165 170 175 Phe Gly His Ser TyrAsn Ser Leu Val Leu Lys Glu Tyr Gly Tyr Thr 180 185 190 Asn Gln Val ValLeu Phe Arg Pro Lys His Leu Gln Ser Lys Phe Glu 195 200 205 Asp Ser GlnVal Val Tyr Asp Gly Asp Lys Ser Asp Lys Gln Glu Leu 210 215 220 Glu GluPhe Val Asn Lys Asn Tyr His Gly Leu Val Gly His Arg Thr 225 230 235 240Thr Asp Asn Thr Asn Gln Phe Ser Pro Pro Leu Ile Val Ser Tyr Tyr 245 250255 Lys Val Asp Tyr Val Lys Asn Thr Lys Gly Thr Asn Tyr Trp Arg Asn 260265 270 Arg Ile Met Lys Val Ala Ser Glu Phe Lys Gly Arg Leu Asn Phe Ala275 280 285 Ile Ser Asn Lys Asp Glu Phe Thr His Glu Leu Ser Glu Tyr GlyPhe 290 295 300 Asn Tyr Val Ala Gly Asp Lys Pro Val Val Ala Ala Arg AsnAla Lys 305 310 315 320 Ser Glu Lys Phe Val Met Glu Gly Glu Phe Ser IlePro Ser Phe Glu 325 330 335 Lys Phe Ile Lys Asp Phe Leu Asp Glu Lys LeuLys Pro Tyr Leu Lys 340 345 350 Ser Glu Pro Ile Pro Glu Lys Asn Glu GluPro Val Lys Val Ala Val 355 360 365 Ala Gln Asn Phe Glu Glu Leu Val ThrLys Ser Asp Lys Asp Val Leu 370 375 380 Ile Glu Phe Tyr Ala Pro Trp CysGly His Cys Lys Lys Leu Ala Pro 385 390 395 400 Val Tyr Asp Glu Leu GlyLys Ala Leu Glu Gly Glu Thr Thr Val Glu 405 410 415 Ile Val Lys Met AspAla Thr Ala Asn Asp Val Pro Ser Pro Tyr Glu 420 425 430 Val His Gly PhePro Thr Leu Tyr Trp Ala Pro Arg Asp Lys Lys Asp 435 440 445 Lys Pro ValArg Tyr Asp Gly Gly Arg Glu Leu Asp Asp Phe Ile Lys 450 455 460 Tyr IleAla Lys His Ser Thr Asp Glu Leu Lys Thr Tyr Asn Arg Asn 465 470 475 480Gly Lys Lys Lys Lys Val Glu Leu 485 <210> SEQ ID NO 15 <211> LENGTH:1498 <212> TYPE: DNA <213> ORGANISM: Hottentotta judiaca <400> SEQUENCE:15 agatctgtaa ggtcatgaat tttggtaatt tattaatctt tttttctttt ttaatagtcg 60tattaggtga agttagagaa gacaatgtat tagttttgaa taaagaaaat tttgatcatt 120caattaaaaa caacaagtat atcttagtag aattttatgc tccatggtgt ggacattgta 180aagcactagc tccagaatat gctaaagctg caaagctgtt gttagaagaa aaatctgaaa 240ttcagttagc aaaaattgat gcaactgaag aaacagaatt agcagagaag cataaagtaa 300aaggttatcc aacaattaaa ttcttccgtg aaggtgatcc tattgattat acaggtggcc 360gtactggtga tgatattgta acttggttga agaaaaaaac tggacctcca gctacattat 420taagtacagt tgatgaagca acaaacttta aagagagtaa agatgtcgta attataggat 480ttttcaagga tcaggaaagt aatcaagcta aagaatattt aaatgcagca tatatgactg 540atgatcatcc atttggtatt acttcagatg aaaatgttta taaacatttt aatgttgaaa 600aggatactat tttcttattt aagaagtttg atgaggggaa gaatgaattc gagggagaat 660ttacaaaaga taacattata aaattcatta aactcaacaa tttaccatta gtaattgaat 720ttagtcaaga gaatgcacag aagatatttg ggggtgacat aaaaatgcat aatcttcttt 780ttattagtaa aaagagtaaa gattttgatg aaatagtgaa aacgtttcgt attgtggcaa 840aagaatataa aaatcagata ttatttgttg ttattaatac tgatgatgaa gacaatgaaa 900aaataactga attctttggt ttaaaaaaag atgagcagcc atcaataaga ttaattaaac 960tagaagaagg aatgtctaaa tataaacctg aaactattga aatttctgaa gaaaatgttc 1020gaaaatttgt taaaggtgtc ttagatggaa cagttaaaca acatctactt tctcaagaac 1080ttcctgaaga ttgggataag catccagtta aagtattagt aaataagaat ttcgatgaag 1140ttgcatttga taaaactaaa gatgttattg tagaattcta tgcaccatgg tgtggtcatt 1200gcaaacagtt agctccaatt tatgaagaac tcggagaaaa atataaaaat cgaaatgata 1260ttattattgc aaaaatggat gcaacagcca atgaattaga acatacaaaa attaacagct 1320ttcctacaat taaattatat aaaaaaggaa ccaatgaagt gatagactat gatggaaaac 1380attcacttga aggacttgtg aattttattg attctggtgg aaaaataaca aaggaacctg 1440aagatgagga taaatcaaaa gaaccagatg ccaaaggaga tgaattatga gcggccgc 1498<210> SEQ ID NO 16 <211> LENGTH: 491 <212> TYPE: PRT <213> ORGANISM:Hottentotta judiaca <400> SEQUENCE: 16 Met Asn Phe Gly Asn Leu Leu IlePhe Phe Ser Phe Leu Ile Val Val 1 5 10 15 Leu Gly Glu Val Arg Glu AspAsn Val Leu Val Leu Asn Lys Glu Asn 20 25 30 Phe Asp His Ser Ile Lys AsnAsn Lys Tyr Ile Leu Val Glu Phe Tyr 35 40 45 Ala Pro Trp Cys Gly His CysLys Ala Leu Ala Pro Glu Tyr Ala Lys 50 55 60 Ala Ala Lys Leu Leu Leu GluGlu Lys Ser Glu Ile Gln Leu Ala Lys 65 70 75 80 Ile Asp Ala Thr Glu GluThr Glu Leu Ala Glu Lys His Lys Val Lys 85 90 95 Gly Tyr Pro Thr Ile LysPhe Phe Arg Glu Gly Asp Pro Ile Asp Tyr 100 105 110 Thr Gly Gly Arg ThrGly Asp Asp Ile Val Thr Trp Leu Lys Lys Lys 115 120 125 Thr Gly Pro ProAla Thr Leu Leu Ser Thr Val Asp Glu Ala Thr Asn 130 135 140 Phe Lys GluSer Lys Asp Val Val Ile Ile Gly Phe Phe Lys Asp Gln 145 150 155 160 GluSer Asn Gln Ala Lys Glu Tyr Leu Asn Ala Ala Tyr Met Thr Asp 165 170 175Asp His Pro Phe Gly Ile Thr Ser Asp Glu Asn Val Tyr Lys His Phe 180 185190 Asn Val Glu Lys Asp Thr Ile Phe Leu Phe Lys Lys Phe Asp Glu Gly 195200 205 Lys Asn Glu Phe Glu Gly Glu Phe Thr Lys Asp Asn Ile Ile Lys Phe210 215 220 Ile Lys Leu Asn Asn Leu Pro Leu Val Ile Glu Phe Ser Gln GluAsn 225 230 235 240 Ala Gln Lys Ile Phe Gly Gly Asp Ile Lys Met His AsnLeu Leu Phe 245 250 255 Ile Ser Lys Lys Ser Lys Asp Phe Asp Glu Ile ValLys Thr Phe Arg 260 265 270 Ile Val Ala Lys Glu Tyr Lys Asn Gln Ile LeuPhe Val Val Ile Asn 275 280 285 Thr Asp Asp Glu Asp Asn Glu Lys Ile ThrGlu Phe Phe Gly Leu Lys 290 295 300 Lys Asp Glu Gln Pro Ser Ile Arg LeuIle Lys Leu Glu Glu Gly Met 305 310 315 320 Ser Lys Tyr Lys Pro Glu ThrIle Glu Ile Ser Glu Glu Asn Val Arg 325 330 335 Lys Phe Val Lys Gly ValLeu Asp Gly Thr Val Lys Gln His Leu Leu 340 345 350 Ser Gln Glu Leu ProGlu Asp Trp Asp Lys His Pro Val Lys Val Leu 355 360 365 Val Asn Lys AsnPhe Asp Glu Val Ala Phe Asp Lys Thr Lys Asp Val 370 375 380 Ile Val GluPhe Tyr Ala Pro Trp Cys Gly His Cys Lys Gln Leu Ala 385 390 395 400 ProIle Tyr Glu Glu Leu Gly Glu Lys Tyr Lys Asn Arg Asn Asp Ile 405 410 415Ile Ile Ala Lys Met Asp Ala Thr Ala Asn Glu Leu Glu His Thr Lys 420 425430 Ile Asn Ser Phe Pro Thr Ile Lys Leu Tyr Lys Lys Gly Thr Asn Glu 435440 445 Val Ile Asp Tyr Asp Gly Lys His Ser Leu Glu Gly Leu Val Asn Phe450 455 460 Ile Asp Ser Gly Gly Lys Ile Thr Lys Glu Pro Glu Asp Glu AspLys 465 470 475 480 Ser Lys Glu Pro Asp Ala Lys Gly Asp Glu Leu 485 490<210> SEQ ID NO 17 <211> LENGTH: 1498 <212> TYPE: DNA <213> ORGANISM:Hottentotta judiaca <400> SEQUENCE: 17 agatctgtaa ggtcatgaat tttggtaatttattaatctt tttttctttt ttaatagtcg 60 tattaggtga agttagagag gacaatgtattagttttgaa taaagaaaat tttgatcatt 120 caattaaaaa caacaagtat atcttagtagaattttatgc tccatggtgt ggacattgta 180 aagcactagc tccagaatat gctaaagctgcaaagctgtt gttagaagaa aaatctgaaa 240 ttcagttagc aaaaattgat gcaactgaagaaacagaatt agcagagaag cataaagtaa 300 aaggttatcc aacaattaaa ttcttccgtgaaggtgatcc tattgattat acaggtggcc 360 gtactggtga tgatattgta acttggttgaagaaaaaaac tggacctcca gctacattat 420 taagtacagt tgatgaagca acaaactttaaagagagtaa agatgtcgta attataggat 480 ttttcaagga tcaggaaagt aatcaagctaaagaatattt aaatgcagca tatatgactg 540 atgatcatcc atttggtatt acttcagatgaaaatgttta taaacatttt aatgttgaaa 600 aggatactat tttcttattt aagaagtttgatgaggggaa gaatgaattc gagggagaat 660 ttacaaaaga taacattata aaattcattaaactcaacaa tttaccatta gtaattgaat 720 ttagtcaaga gaatgcacag aagatatttgggggtgacat aaaaatgcat aatcttcttt 780 ttattagtaa aaagagtaaa gattttgatgaaatagtgaa aacgtttcgt attgtggcaa 840 aagaatataa aaatcagata ttatttgttgttattaatac tgatgatgaa ggcaatggac 900 aaataactga attctttggt ttaaaaaaggatgagcagcc atcaataaga ttaattaaac 960 tagaagaagg aatgtctaaa tataaacctgaaactattga aatttctgaa gaaaatgttc 1020 gaaaatttgt taaaggtgtc ttagatggaacagttaaaca acatctactt tctcaagaac 1080 ttcctgaaga ttgggataag catccagttaaagtattagt aaataagaat ttcgatgaag 1140 ttgcatttga taaaactaaa gatgttattgtagaattcta tgcaccatgg tgtggtcatt 1200 gcaaacagtt agctccaatt tatgaagaactcggagaaaa atataaaaat cgaaatgata 1260 ttattattgc aaaaatggat gcaacagccaatgaattaga acatacaaaa attaacagct 1320 ttcctacaat taaattatat aaaaaaggaaccaatgaagt gatagactat gatggaaaac 1380 attcacttga aggacttgtg aattttattgattctggtgg aaaaataaca aaggaacctg 1440 aagatgagga taaatcaaaa gaaccagatgccaaaagaga tgaattatga gcggccgc 1498 <210> SEQ ID NO 18 <211> LENGTH: 491<212> TYPE: PRT <213> ORGANISM: Hottentotta judiaca <400> SEQUENCE: 18Met Asn Phe Gly Asn Leu Leu Ile Phe Phe Ser Phe Leu Ile Val Val 1 5 1015 Leu Gly Glu Val Arg Glu Asp Asn Val Leu Val Leu Asn Lys Glu Asn 20 2530 Phe Asp His Ser Ile Lys Asn Asn Lys Tyr Ile Leu Val Glu Phe Tyr 35 4045 Ala Pro Trp Cys Gly His Cys Lys Ala Leu Ala Pro Glu Tyr Ala Lys 50 5560 Ala Ala Lys Leu Leu Leu Glu Glu Lys Ser Glu Ile Gln Leu Ala Lys 65 7075 80 Ile Asp Ala Thr Glu Glu Thr Glu Leu Ala Glu Lys His Lys Val Lys 8590 95 Gly Tyr Pro Thr Ile Lys Phe Phe Arg Glu Gly Asp Pro Ile Asp Tyr100 105 110 Thr Gly Gly Arg Thr Gly Asp Asp Ile Val Thr Trp Leu Lys LysLys 115 120 125 Thr Gly Pro Pro Ala Thr Leu Leu Ser Thr Val Asp Glu AlaThr Asn 130 135 140 Phe Lys Glu Ser Lys Asp Val Val Ile Ile Gly Phe PheLys Asp Gln 145 150 155 160 Glu Ser Asn Gln Ala Lys Glu Tyr Leu Asn AlaAla Tyr Met Thr Asp 165 170 175 Asp His Pro Phe Gly Ile Thr Ser Asp GluAsn Val Tyr Lys His Phe 180 185 190 Asn Val Glu Lys Asp Thr Ile Phe LeuPhe Lys Lys Phe Asp Glu Gly 195 200 205 Lys Asn Glu Phe Glu Gly Glu PheThr Lys Asp Asn Ile Ile Lys Phe 210 215 220 Ile Lys Leu Asn Asn Leu ProLeu Val Ile Glu Phe Ser Gln Glu Asn 225 230 235 240 Ala Gln Lys Ile PheGly Gly Asp Ile Lys Met His Asn Leu Leu Phe 245 250 255 Ile Ser Lys LysSer Lys Asp Phe Asp Glu Ile Val Lys Thr Phe Arg 260 265 270 Ile Val AlaLys Glu Tyr Lys Asn Gln Ile Leu Phe Val Val Ile Asn 275 280 285 Thr AspAsp Glu Gly Asn Gly Gln Ile Thr Glu Phe Phe Gly Leu Lys 290 295 300 LysAsp Glu Gln Pro Ser Ile Arg Leu Ile Lys Leu Glu Glu Gly Met 305 310 315320 Ser Lys Tyr Lys Pro Glu Thr Ile Glu Ile Ser Glu Glu Asn Val Arg 325330 335 Lys Phe Val Lys Gly Val Leu Asp Gly Thr Val Lys Gln His Leu Leu340 345 350 Ser Gln Glu Leu Pro Glu Asp Trp Asp Lys His Pro Val Lys ValLeu 355 360 365 Val Asn Lys Asn Phe Asp Glu Val Ala Phe Asp Lys Thr LysAsp Val 370 375 380 Ile Val Glu Phe Tyr Ala Pro Trp Cys Gly His Cys LysGln Leu Ala 385 390 395 400 Pro Ile Tyr Glu Glu Leu Gly Glu Lys Tyr LysAsn Arg Asn Asp Ile 405 410 415 Ile Ile Ala Lys Met Asp Ala Thr Ala AsnGlu Leu Glu His Thr Lys 420 425 430 Ile Asn Ser Phe Pro Thr Ile Lys LeuTyr Lys Lys Gly Thr Asn Glu 435 440 445 Val Ile Asp Tyr Asp Gly Lys HisSer Leu Glu Gly Leu Val Asn Phe 450 455 460 Ile Asp Ser Gly Gly Lys IleThr Lys Glu Pro Glu Asp Glu Asp Lys 465 470 475 480 Ser Lys Glu Pro AspAla Lys Arg Asp Glu Leu 485 490 <210> SEQ ID NO 19 <211> LENGTH: 802<212> TYPE: DNA <213> ORGANISM: Hottentotta judiaca <400> SEQUENCE: 19gcacgaggga acatgggatg ttacttgttg gtgttgttga tatttttatt ctttctacga 60gatagtcaaa gtagcagtga tttatacacg gataactcga taaaatatga cgaggaagga 120tttaggagga atataggaaa tatagtgcat tttgttaaat tttacgcccc ttggtgtgga 180cattgtaaaa gattagcacc aatttgggat gaattagcag agaaatataa taaacctgga 240gaacagaagc ttgttattgc taaaattgat tgtacaactg aaactgctct ttgttctgaa 300caaggaatta ctggttatcc cacattaaag ttttttaaga aaggtacaac tgaaggacat 360aaatatagag gtccacgtga cattacttct ttagaagctt ttattgccaa tagcttagga 420cacgaagagg ctattaaaaa atctcctgaa cctccaaaat tcataaatga aattattcag 480ttaagtgaca atacttttca taaatttgta gcaaaaggac ttcattttgt taaattttat 540gctccttggt gtggtcactg tcagaaactt gttcccattt ggaaagaatt ggcaaatagc 600tttaaatttg atacatccat aaaaatatct gagattgatt gcactacaca acatttagta 660tgtaatgaat ttgaagttaa agcatatcca actttattgt ggattgttga tggtaaaaag 720attgaaaagt atgaaggaat gagatcccat gaagaactaa aattatttat taataaaatg 780aaagaaaaaa aaaaaaaaaa aa 802 <210> SEQ ID NO 20 <211> LENGTH: 244 <212>TYPE: PRT <213> ORGANISM: Hottentotta judiaca <400> SEQUENCE: 20 Met GlyCys Tyr Leu Leu Val Leu Leu Ile Phe Leu Phe Phe Leu Arg 1 5 10 15 AspSer Gln Ser Ser Ser Asp Leu Tyr Thr Asp Asn Ser Ile Lys Tyr 20 25 30 AspGlu Glu Gly Phe Arg Arg Asn Ile Gly Asn Ile Val His Phe Val 35 40 45 LysPhe Tyr Ala Pro Trp Cys Gly His Cys Lys Arg Leu Ala Pro Ile 50 55 60 TrpAsp Glu Leu Ala Glu Lys Tyr Asn Lys Pro Gly Glu Gln Lys Leu 65 70 75 80Val Ile Ala Lys Ile Asp Cys Thr Thr Glu Thr Ala Leu Cys Ser Glu 85 90 95Gln Gly Ile Thr Gly Tyr Pro Thr Leu Lys Phe Phe Lys Lys Gly Thr 100 105110 Thr Glu Gly His Lys Tyr Arg Gly Pro Arg Asp Ile Thr Ser Leu Glu 115120 125 Ala Phe Ile Ala Asn Ser Leu Gly His Glu Glu Ala Ile Lys Lys Ser130 135 140 Pro Glu Pro Pro Lys Phe Ile Asn Glu Ile Ile Gln Leu Ser AspAsn 145 150 155 160 Thr Phe His Lys Phe Val Ala Lys Gly Leu His Phe ValLys Phe Tyr 165 170 175 Ala Pro Trp Cys Gly His Cys Gln Lys Leu Val ProIle Trp Lys Glu 180 185 190 Leu Ala Asn Ser Phe Lys Phe Asp Thr Ser IleLys Ile Ser Glu Ile 195 200 205 Asp Cys Thr Thr Gln His Leu Val Cys AsnGlu Phe Glu Val Lys Ala 210 215 220 Tyr Pro Thr Leu Leu Trp Ile Val AspGly Lys Lys Ile Glu Lys Tyr 225 230 235 240 Glu Gly Met Arg <210> SEQ IDNO 21 <211> LENGTH: 1495 <212> TYPE: DNA <213> ORGANISM: Heliothusvirescens] <400> SEQUENCE: 21 gcacgagaaa cccgatcgtt catcagaaaagtcggactct gacgtgatca cgctgacgga 60 tgagaacttc aagaagctgg tgctggacagcgaagacctg tggctggttg agttctacgc 120 gccctggtgc ggtcactgca agaatttgaagccccagtgg gccaaggctg ccaaagaact 180 taagggcaag gtgaaactcg gagcattagacgcgacagtc caccaagcga tggcttcccg 240 ctaccaagtg caaggctacc ccaccatcaagctgttccca tctggcaaga agtccagtga 300 ctccgcagag gactacaatg gaggcaggaccgccagcgac atcgtgactt atgctcttga 360 caagctcgct gaaaacgtgc ccgctcctgagatcgttcag gttatcgacg aagcgtcaat 420 gcaggcgtgc agtgaaaaac cgctgtgcgtggtatcggtt ctgccgcaca tcttggactg 480 caacgcggcc tgtcgcaacg aatacctagcgatactcgca cgactcggtg acaagtacaa 540 gagcaagatg tggggatggg tgtgggccgaagctggcgcg cagatatctt tggaagagtc 600 gctggagctg ggcggtttcg gttaccccgccatggctgtc gtcaacgcta agaaactcaa 660 gttctcaacc ctcaggggat ccttctccgagactggcatc aatgaattcc ttagggatct 720 atcattcggt cgcggacaga ctgccccagtgaagggcgca gagatgccga agatcgtgtc 780 caccgacccc tgggacggca aggacggtgaactgccacaa gaagaggaca ttgacctctc 840 tgacgtagac ctcgagaagg acgagttataagtgcagcag ccatgttgct aacagtctgg 900 actttataaa acccaacggt tgagtgttctgtaacaagta cgcttctaca caaaatcata 960 tcagtaaaaa tctctgattt taaacttaagaaagtgatac aagttcaagc atttaacagt 1020 ttaggttact atttattttc accagtgagctagtaacttt gtacctaata atatggttca 1080 gtttaaaatt atgctgtttt aaatatcgaaggagaagctt aattccatca catactatga 1140 attttatttt ctgaaacatt tttaggtgtttgataatcac aatttagtac cagccatata 1200 tttcgtgtgt agctcggcgc gagcgagtggtgcaacgact gatctttttg aatcattgtt 1260 atttgtatgt atatccttca tagtcataaaattgataaca caaactgata cttaatttta 1320 gttggattag acattaattg gagtgtacattagctaaacg ccaatcttcc aatattatgt 1380 ttaattttgg taagcattta ttgttgtgaggagatttgga taattttatg aattgataaa 1440 tcgctaataa atttttaata aaaaaaaaaaaaaaagagag agagagagaa ctagt 1495 <210> SEQ ID NO 22 <211> LENGTH: 289<212> TYPE: PRT <213> ORGANISM: Heliothus virescens] <400> SEQUENCE: 22His Glu Lys Pro Asp Arg Ser Ser Glu Lys Ser Asp Ser Asp Val Ile 1 5 1015 Thr Leu Thr Asp Glu Asn Phe Lys Lys Leu Val Leu Asp Ser Glu Asp 20 2530 Leu Trp Leu Val Glu Phe Tyr Ala Pro Trp Cys Gly His Cys Lys Asn 35 4045 Leu Lys Pro Gln Trp Ala Lys Ala Ala Lys Glu Leu Lys Gly Lys Val 50 5560 Lys Leu Gly Ala Leu Asp Ala Thr Val His Gln Ala Met Ala Ser Arg 65 7075 80 Tyr Gln Val Gln Gly Tyr Pro Thr Ile Lys Leu Phe Pro Ser Gly Lys 8590 95 Lys Ser Ser Asp Ser Ala Glu Asp Tyr Asn Gly Gly Arg Thr Ala Ser100 105 110 Asp Ile Val Thr Tyr Ala Leu Asp Lys Leu Ala Glu Asn Val ProAla 115 120 125 Pro Glu Ile Val Gln Val Ile Asp Glu Ala Ser Met Gln AlaCys Ser 130 135 140 Glu Lys Pro Leu Cys Val Val Ser Val Leu Pro His IleLeu Asp Cys 145 150 155 160 Asn Ala Ala Cys Arg Asn Glu Tyr Leu Ala IleLeu Ala Arg Leu Gly 165 170 175 Asp Lys Tyr Lys Ser Lys Met Trp Gly TrpVal Trp Ala Glu Ala Gly 180 185 190 Ala Gln Ile Ser Leu Glu Glu Ser LeuGlu Leu Gly Gly Phe Gly Tyr 195 200 205 Pro Ala Met Ala Val Val Asn AlaLys Lys Leu Lys Phe Ser Thr Leu 210 215 220 Arg Gly Ser Phe Ser Glu ThrGly Ile Asn Glu Phe Leu Arg Asp Leu 225 230 235 240 Ser Phe Gly Arg GlyGln Thr Ala Pro Val Lys Gly Ala Glu Met Pro 245 250 255 Lys Ile Val SerThr Asp Pro Trp Asp Gly Lys Asp Gly Glu Leu Pro 260 265 270 Gln Glu GluAsp Ile Asp Leu Ser Asp Val Asp Leu Glu Lys Asp Glu 275 280 285 Leu<210> SEQ ID NO 23 <211> LENGTH: 473 <212> TYPE: DNA <213> ORGANISM:Vaejovis carolinianus <220> FEATURE: <221> NAME/KEY: unsure <222>LOCATION: (408) <221> NAME/KEY: unsure <222> LOCATION: (442) <221>NAME/KEY: unsure <222> LOCATION: (444) <400> SEQUENCE: 23 gttgtcatctgtttttagtt ttaatgttgc aatatatgct agtagtgact tatacgttga 60 caattcgttaaaatatgatg aagatggttt tagggaaaat gtaggaaaat tgacgctttt 120 tgtgaaattctacgcccctt ggtgtggaca ttgtaaaaga ttggctccta cttgggacga 180 actagctgaaaaatataata ttcaaccaga aaaacaacag gtcataatag ctaagattga 240 ctgtacatcagagacagctc tttgttctga gcaaggaata acaggttatc caacattaaa 300 gttttttaagaaaggtgaaa ctgaaggaac aaaatacagg ggaccaagag acatcacatc 360 tttagaagcttttattgcta acagcttggg caaagaagag gctgtggnaa gatcttaaac 420 caccaagaaccaagtaaatg gncnaataag aattaactga tgaaacattc cac 473 <210> SEQ ID NO 24<211> LENGTH: 343 <212> TYPE: PRT <213> ORGANISM: Vaejovis carolinianus<400> SEQUENCE: 24 Thr Arg Leu Ser Ser Val Phe Ser Phe Asn Val Ala IleTyr Ala Ser 1 5 10 15 Ser Asp Leu Tyr Val Asp Asn Ser Leu Lys Tyr AspGlu Asp Gly Phe 20 25 30 Arg Glu Asn Val Gly Lys Leu Thr Leu Phe Val LysPhe Tyr Ala Pro 35 40 45 Trp Cys Gly His Cys Lys Arg Leu Ala Pro Thr TrpAsp Glu Leu Ala 50 55 60 Glu Lys Tyr Asn Ile Gln Pro Glu Lys Gln Gln ValIle Ile Ala Lys 65 70 75 80 Ile Asp Cys Thr Ser Glu Thr Ala Leu Cys SerGlu Gln Gly Ile Thr 85 90 95 Gly Tyr Pro Thr Leu Lys Phe Phe Lys Lys GlyGlu Thr Glu Gly Thr 100 105 110 Lys Tyr Arg Gly Pro Arg Asp Ile Thr SerLeu Glu Ala Phe Ile Ala 115 120 125 Asn Ser Leu Gly Lys Glu Glu Ala ValGlu Asp Leu Lys Pro Pro Glu 130 135 140 Pro Val Asn Gly Leu Ile Glu LeuThr Asp Glu Thr Phe His Lys Thr 145 150 155 160 Ile Glu Arg Gly Tyr HisPhe Val Lys Phe Tyr Ala Pro Trp Cys Gly 165 170 175 His Cys Gln Lys LeuAla Pro Val Trp Gln Gln Leu Ala Asn Ser Phe 180 185 190 Gln His Asp LeuSer Val Lys Ile Leu Lys Ile Asp Cys Thr Ala His 195 200 205 Arg Leu SerCys Asn Glu Phe Glu Val Lys Ala Tyr Pro Thr Leu Leu 210 215 220 Trp IleVal Asp Gly Lys Lys Val Glu Ile Tyr Gln Gly Ser Arg Thr 225 230 235 240His Glu Asp Leu Lys Leu Phe Val Asp Lys Met Arg Arg Gln Glu His 245 250255 Glu Thr Asp Ser Gly Gly Glu His Gly Lys Ile Pro Glu Ser Leu Pro 260265 270 Lys Pro Glu Ala Pro Val Ala Gln Leu Val Ala Ser Asn Phe Glu Asp275 280 285 Ser Ile Lys Asn Gly Val Thr Phe Val Lys Phe Phe Ala Pro TrpCys 290 295 300 Gly His Cys Arg Lys Leu Ala Pro Ile Trp Asp Glu Leu SerTrp Glu 305 310 315 320 Phe Ile Asp Asn Glu Asn Gly Lys Ile Ala Gln ValAsp Cys Ser Ser 325 330 335 Gln Glu Ser Leu Cys Ser Lys 340 <210> SEQ IDNO 25 <211> LENGTH: 2209 <212> TYPE: DNA <213> ORGANISM: Spodopterafrugiperda <400> SEQUENCE: 25 gcacgagata agttgtgcga tctcattaaaaaatagtcgg tgtttttata gtttttaaat 60 taagtagaat ataatcaaca caatgttacacacctatttc ctgggtattt tattgtgtgt 120 ggggtctggg ttggcgttgt atgactccagttcggacgtg gtggacctga cacccgacaa 180 tttctatcaa ctagtcacag atagagatgatgtatggttg gtggaattct acgcgccgtg 240 gtgcggtcac tgcaagaact tggtgcctgaatacaagaaa gcggccaaag ctctgaaggg 300 tattgttaaa gtgggagcta tagacgcagacaagcacaga agcttcgcaa aggactatgg 360 agtgtctggc ttccccacaa ttaagatctttacgggtcgt aaacatgttc catacaaggg 420 cgcaaggtca gctgatgctt tcgttgatgctgctctaagt gcagtgaaga gcaaggctta 480 tgagagactt ggaaagagat ccgatgactcatcacacaag tcatccgact ctgacgtgat 540 cacgctgaca gacgacaact tcaagaaactggtgttggac agcgatgacc tgtggttggt 600 ggagttcttc gccccatggt gcggacactgcaagaacctc gagccacact gggctaaggc 660 agctactgaa cttaagggca aggtgaaagtgggagctctc gacgctactg ttcaccagga 720 gatggcaggc cgcttccaag tccaaggctacccaaccatc aagtacttcc catcaggcaa 780 gaagacctac gactctgctg aggactacaacggaggcagg acatccagtg acatcgtgtc 840 attcgccctc gaaaagctgg ctgagaatgtacccgctcct gagattattc aggttgtcaa 900 cgaagcaaca atgcaggcgt gcagcgagaagccgctgtgt gtggtatcgg tgctgcctca 960 catcttcgac tgtaacgcgg cctgccgcaacgactacctc gccatactcg cccgtctcgg 1020 agacaagtac aagaacaaga tgtggggatgggtatgggct gaagctggtg cccaacttgg 1080 tctagaagag tctctggaac tcggcggcttcggctacccc gccatggctg tggtcaacgc 1140 taagaaactc aagttctcaa cacttaggggatccttctcc gaaactggta tcaacgagtt 1200 ccttagggac ctgtcattcg gtcgcggccagactgcgcca gtcagaggcg ctgagatgcc 1260 caagatagtg tcgacagacg cttgggacggcaaggacggt gaactgcccc aggaagagga 1320 catagaccta tcagacgtgg accttgagaaggacgagtta taagtgcaaa cagtgactta 1380 gacaagtgta gctaggcggg aatgtcctttgtactgagat caacactcaa tacatactaa 1440 aaaaaacatc ataaaagtta tttaatcacttctagaaggt tccaaagcct agctacgcta 1500 caagatactc gtatctcata aactgtaccagtggttgagt gcatttgaag ttatctgcag 1560 tgagaacaca caaataaatt catatcaaatctctgcttta tagaaagatt gcaggttcga 1620 gcatttgttc atagtatttt attgaagtgagccagtgaat agttttactg ttagataaat 1680 taatatgtaa catagtttga tactatgctgcaatacagga actatttatt ccaagctgga 1740 ttaagcatga gttagggtct gtagcaaaatctacaggcca aaataattaa ggcaatggtg 1800 attttagtta tgcaatttct actctagctacatggttaat ccagccctga ttccatcaca 1860 tgttatttac tttattttct gatatatttagcgtatctga agatcacaaa ttgaaaacgt 1920 aatgtttgga gctgataagc tcggctctagcgagcaatgt aatgactgat gtttctgaat 1980 cattttactt attatttctt attcatgatacaaaatataa aaggactgat acctttttta 2040 gttaatgatt ggaattaatt ctccatcagccattcttcca ataatattgg tttagcgtgg 2100 taagctttta ttgtaatcgt tgtgaggagatattttggat aattttatga attgtaaaat 2160 cgctaataaa tttttaatat aattaagtcaaaaaaaaaaa aaaaaaaaa 2209 <210> SEQ ID NO 26 <211> LENGTH: 426 <212>TYPE: PRT <213> ORGANISM: Spodoptera frugiperda <400> SEQUENCE: 26 MetLeu His Thr Tyr Phe Leu Gly Ile Leu Leu Cys Val Gly Ser Gly 1 5 10 15Leu Ala Leu Tyr Asp Ser Ser Ser Asp Val Val Asp Leu Thr Pro Asp 20 25 30Asn Phe Tyr Gln Leu Val Thr Asp Arg Asp Asp Val Trp Leu Val Glu 35 40 45Phe Tyr Ala Pro Trp Cys Gly His Cys Lys Asn Leu Val Pro Glu Tyr 50 55 60Lys Lys Ala Ala Lys Ala Leu Lys Gly Ile Val Lys Val Gly Ala Ile 65 70 7580 Asp Ala Asp Lys His Arg Ser Phe Ala Lys Asp Tyr Gly Val Ser Gly 85 9095 Phe Pro Thr Ile Lys Ile Phe Thr Gly Arg Lys His Val Pro Tyr Lys 100105 110 Gly Ala Arg Ser Ala Asp Ala Phe Val Asp Ala Ala Leu Ser Ala Val115 120 125 Lys Ser Lys Ala Tyr Glu Arg Leu Gly Lys Arg Ser Asp Asp SerSer 130 135 140 His Lys Ser Ser Asp Ser Asp Val Ile Thr Leu Thr Asp AspAsn Phe 145 150 155 160 Lys Lys Leu Val Leu Asp Ser Asp Asp Leu Trp LeuVal Glu Phe Phe 165 170 175 Ala Pro Trp Cys Gly His Cys Lys Asn Leu GluPro His Trp Ala Lys 180 185 190 Ala Ala Thr Glu Leu Lys Gly Lys Val LysVal Gly Ala Leu Asp Ala 195 200 205 Thr Val His Gln Glu Met Ala Gly ArgPhe Gln Val Gln Gly Tyr Pro 210 215 220 Thr Ile Lys Tyr Phe Pro Ser GlyLys Lys Thr Tyr Asp Ser Ala Glu 225 230 235 240 Asp Tyr Asn Gly Gly ArgThr Ser Ser Asp Ile Val Ser Phe Ala Leu 245 250 255 Glu Lys Leu Ala GluAsn Val Pro Ala Pro Glu Ile Ile Gln Val Val 260 265 270 Asn Glu Ala ThrMet Gln Ala Cys Ser Glu Lys Pro Leu Cys Val Val 275 280 285 Ser Val LeuPro His Ile Phe Asp Cys Asn Ala Ala Cys Arg Asn Asp 290 295 300 Tyr LeuAla Ile Leu Ala Arg Leu Gly Asp Lys Tyr Lys Asn Lys Met 305 310 315 320Trp Gly Trp Val Trp Ala Glu Ala Gly Ala Gln Leu Gly Leu Glu Glu 325 330335 Ser Leu Glu Leu Gly Gly Phe Gly Tyr Pro Ala Met Ala Val Val Asn 340345 350 Ala Lys Lys Leu Lys Phe Ser Thr Leu Arg Gly Ser Phe Ser Glu Thr355 360 365 Gly Ile Asn Glu Phe Leu Arg Asp Leu Ser Phe Gly Arg Gly GlnThr 370 375 380 Ala Pro Val Arg Gly Ala Glu Met Pro Lys Ile Val Ser ThrAsp Ala 385 390 395 400 Trp Asp Gly Lys Asp Gly Glu Leu Pro Gln Glu GluAsp Ile Asp Leu 405 410 415 Ser Asp Val Asp Leu Glu Lys Asp Glu Leu 420425 <210> SEQ ID NO 27 <211> LENGTH: 496 <212> TYPE: PRT <213> ORGANISM:Drosophila melanogaster <400> SEQUENCE: 27 Met Lys Phe Leu Ile Cys AlaLeu Phe Leu Ala Ala Ser Tyr Val Ala 1 5 10 15 Ala Ser Ala Glu Ala GluVal Lys Val Glu Glu Gly Val Leu Val Ala 20 25 30 Thr Val Asp Asn Phe LysGln Leu Ile Ala Asp Asn Glu Phe Val Leu 35 40 45 Val Glu Phe Tyr Ala ProTrp Cys Gly His Cys Lys Ala Leu Ala Pro 50 55 60 Glu Tyr Ala Lys Ala AlaGln Gln Leu Ala Glu Lys Glu Ser Pro Ile 65 70 75 80 Lys Leu Ala Lys ValAsp Ala Thr Val Glu Gly Glu Leu Ala Glu Gln 85 90 95 Tyr Ala Val Arg GlyTyr Pro Thr Leu Lys Phe Phe Arg Ser Gly Ser 100 105 110 Pro Val Glu TyrSer Gly Gly Arg Gln Ala Ala Asp Ile Ile Ala Trp 115 120 125 Val Thr LysLys Thr Gly Pro Pro Ala Lys Asp Leu Thr Ser Val Ala 130 135 140 Asp AlaGlu Gln Phe Leu Lys Asp Asn Glu Ile Ala Ile Ile Gly Phe 145 150 155 160Phe Lys Asp Leu Glu Ser Glu Glu Ala Lys Thr Phe Thr Lys Val Ala 165 170175 Asn Ala Leu Asp Ser Phe Val Phe Gly Val Ser Ser Asn Ala Asp Val 180185 190 Ile Ala Lys Tyr Glu Ala Lys Asp Asn Gly Val Val Leu Phe Lys Pro195 200 205 Phe Asp Asp Lys Lys Ser Val Phe Glu Gly Glu Leu Asn Glu GluAsn 210 215 220 Leu Lys Lys Phe Ala Gln Val Gln Ser Leu Pro Leu Ile ValAsp Phe 225 230 235 240 Asn His Glu Ser Ala Ser Lys Ile Phe Gly Gly SerIle Lys Ser His 245 250 255 Leu Leu Phe Phe Val Ser Arg Glu Gly Gly HisIle Glu Lys Tyr Val 260 265 270 Asp Pro Leu Lys Glu Ile Ala Lys Lys TyrArg Asp Asp Ile Leu Phe 275 280 285 Val Thr Ile Ser Ser Asp Glu Glu AspHis Thr Arg Ile Phe Glu Phe 290 295 300 Phe Gly Met Asn Lys Glu Glu ValPro Thr Ile Arg Leu Ile Lys Leu 305 310 315 320 Glu Glu Asp Met Ala LysTyr Lys Pro Glu Ser Asp Asp Leu Ser Ala 325 330 335 Glu Thr Ile Glu AlaPhe Leu Lys Lys Phe Leu Asp Gly Lys Leu Lys 340 345 350 Gln His Leu LeuSer Gln Glu Leu Pro Glu Asp Trp Asp Lys Asn Pro 355 360 365 Val Lys ValLeu Val Ser Ser Asn Phe Glu Ser Val Ala Leu Asp Lys 370 375 380 Ser LysSer Val Leu Val Glu Phe Tyr Ala Pro Trp Cys Gly His Cys 385 390 395 400Lys Gln Leu Ala Pro Ile Tyr Asp Gln Leu Ala Glu Lys Tyr Lys Asp 405 410415 Asn Glu Asp Ile Val Ile Ala Lys Met Asp Ser Thr Ala Asn Glu Leu 420425 430 Glu Ser Ile Lys Ile Ser Ser Phe Pro Thr Ile Lys Tyr Phe Arg Lys435 440 445 Glu Asp Asn Lys Val Ile Asp Phe Asn Leu Asp Arg Thr Leu AspAsp 450 455 460 Phe Val Lys Phe Leu Asp Ala Asn Gly Glu Val Ala Asp SerGlu Pro 465 470 475 480 Val Glu Glu Thr Glu Glu Glu Glu Glu Ala Pro LysLys Asp Glu Leu 485 490 495 <210> SEQ ID NO 28 <211> LENGTH: 515 <212>TYPE: PRT <213> ORGANISM: Gallus gallus <400> SEQUENCE: 28 Met Ala ValVal Arg Val Arg Ala Ile Val Ala Leu Leu Cys Leu Val 1 5 10 15 Ala AlaLeu Gly Leu Ala Glu Pro Leu Glu Glu Glu Asp Gly Val Leu 20 25 30 Val LeuArg Ala Ala Asn Phe Glu Gln Ala Leu Ala Ala His Arg His 35 40 45 Leu LeuVal Glu Phe Tyr Ala Pro Trp Cys Gly His Cys Lys Ala Leu 50 55 60 Ala ProGlu Tyr Ala Lys Ala Ala Ala Gln Leu Lys Ala Glu Gly Ser 65 70 75 80 GluIle Arg Leu Ala Lys Val Asp Ala Thr Glu Glu Ala Glu Leu Ala 85 90 95 GlnGln Phe Gly Val Arg Gly Tyr Pro Thr Ile Lys Phe Phe Arg Asn 100 105 110Gly Asp Lys Ala Ala Pro Arg Glu Tyr Thr Ala Gly Arg Glu Ala Asp 115 120125 Asp Ile Val Ser Trp Leu Lys Lys Arg Thr Gly Pro Ala Ala Thr Thr 130135 140 Leu Thr Asp Ala Ala Ala Ala Glu Thr Leu Val Asp Ser Ser Glu Val145 150 155 160 Val Val Ile Gly Phe Phe Lys Asp Val Thr Ser Asp Ala AlaLys Glu 165 170 175 Phe Leu Leu Ala Ala Glu Ser Val Asp Asp Ile Pro PheGly Ile Ser 180 185 190 Ser Ser Ala Asp Val Phe Ser Lys Tyr Gln Leu SerGln Asp Gly Val 195 200 205 Val Leu Phe Lys Lys Phe Asp Glu Gly Arg AsnAsn Phe Glu Gly Asp 210 215 220 Leu Thr Lys Asp Asn Leu Leu Asn Phe IleLys Ser Asn Gln Leu Pro 225 230 235 240 Leu Val Ile Glu Phe Thr Glu GlnThr Ala Pro Lys Ile Phe Gly Gly 245 250 255 Glu Ile Lys Thr His Ile LeuLeu Phe Leu Pro Lys Ser Val Ser Asp 260 265 270 Tyr Glu Gly Lys Leu AspAsn Phe Lys Thr Ala Ala Gly Asn Phe Lys 275 280 285 Gly Lys Ile Leu PheIle Phe Ile Asp Ser Asp His Ser Asp Asn Gln 290 295 300 Arg Ile Leu GluPhe Phe Gly Leu Lys Lys Glu Glu Cys Pro Ala Val 305 310 315 320 Arg LeuIle Thr Leu Glu Glu Glu Met Thr Lys Tyr Lys Pro Glu Ser 325 330 335 AspAsp Leu Thr Ala Asp Lys Ile Lys Glu Phe Cys Asn Lys Phe Leu 340 345 350Glu Gly Lys Ile Lys Pro His Leu Met Ser Gln Asp Leu Pro Glu Asp 355 360365 Trp Asp Lys Gln Pro Val Lys Val Leu Val Gly Lys Asn Phe Glu Glu 370375 380 Val Ala Phe Asp Glu Asn Lys Asn Val Phe Val Glu Phe Tyr Ala Pro385 390 395 400 Trp Cys Gly His Cys Lys Gln Leu Ala Pro Ile Trp Asp LysLeu Gly 405 410 415 Glu Thr Tyr Arg Asp His Glu Asn Ile Val Ile Ala LysMet Asp Ser 420 425 430 Thr Ala Asn Glu Val Glu Ala Val Lys Ile His SerPhe Pro Thr Leu 435 440 445 Lys Phe Phe Pro Ala Gly Ser Gly Arg Asn ValIle Asp Tyr Asn Gly 450 455 460 Glu Arg Thr Leu Glu Gly Phe Lys Lys PheLeu Glu Ser Gly Gly Gln 465 470 475 480 Asp Gly Ala Ala Ala Asp Asp AspLeu Glu Asp Leu Glu Thr Asp Glu 485 490 495 Glu Thr Asp Leu Glu Glu GlyAsp Asp Asp Glu Gln Lys Ile Gln Lys 500 505 510 Asp Glu Leu 515 <210>SEQ ID NO 29 <211> LENGTH: 489 <212> TYPE: PRT <213> ORGANISM:Drosophila melanogaster <400> SEQUENCE: 29 Met Met Trp Arg Leu Ala GlyVal Leu Leu Leu Gly Phe Ile Ala Ile 1 5 10 15 Ser Ser Gly Ala Asp GluAsp Val Leu Glu Leu Gly Asp Asp Asp Phe 20 25 30 Ala Thr Thr Leu Lys GlnHis Glu Thr Thr Leu Val Met Phe Tyr Ala 35 40 45 Pro Trp Cys Gly His CysLys Arg Leu Lys Pro Glu Tyr Ala Lys Ala 50 55 60 Ala Glu Ile Val Lys AspAsp Asp Pro Pro Ile Lys Leu Ala Lys Val 65 70 75 80 Asp Cys Thr Glu AlaGly Lys Glu Thr Cys Ser Lys Tyr Ser Val Ser 85 90 95 Gly Tyr Pro Thr LeuLys Ile Phe Arg Gln Asp Glu Val Ser Gln Asp 100 105 110 Tyr Asn Gly ProArg Asp Ser Ser Gly Ile Ala Lys Tyr Met Arg Ala 115 120 125 Gln Val GlyPro Ala Ser Lys Thr Val Arg Thr Val Ala Glu Leu Lys 130 135 140 Lys PheLeu Asp Thr Lys Asp Thr Thr Leu Phe Gly Tyr Phe Ser Asp 145 150 155 160Ser Asp Ser Lys Leu Ala Lys Ile Phe Leu Lys Phe Ala Asp Lys Asn 165 170175 Arg Glu Lys Tyr Arg Phe Gly His Ser Ser Glu Lys Glu Val Leu Asp 180185 190 Lys Gln Gly Glu Thr Asp Lys Ile Val Leu Ile Arg Ala Pro His Leu195 200 205 Ser Asn Lys Phe Glu Ser Ser Ser Ile Lys Phe Glu Gly Ser SerGlu 210 215 220 Ser Asp Leu Ser Thr Phe Val Lys Glu Asn Phe His Gly LeuVal Gly 225 230 235 240 His Arg Thr Gln Asp Ser Val Lys Asp Phe Gln AsnPro Leu Ile Thr 245 250 255 Ala Tyr Tyr Ser Val Asp Tyr Gln Lys Asn ProLys Gly Thr Asn Tyr 260 265 270 Trp Arg Asn Arg Val Leu Lys Val Ala LysGlu Phe Val Gly Gln Ile 275 280 285 Asn Phe Ala Ile Ala Ser Lys Asp AspPhe Gln His Glu Leu Asn Glu 290 295 300 Tyr Gly Tyr Asp Phe Val Gly AspLys Pro Val Val Leu Ala Arg Asp 305 310 315 320 Glu Lys Asn Leu Lys TyrAla Leu Lys Asp Glu Phe Ser Val Glu Asn 325 330 335 Leu Gln Asp Phe ValGlu Lys Leu Leu Ala Asn Glu Leu Glu Pro Tyr 340 345 350 Ile Lys Ser GluPro Ile Pro Glu Ser Asn Asp Ala Pro Val Lys Val 355 360 365 Ala Val AlaLys Asn Phe Asp Asp Leu Val Ile Asn Asn Gly Lys Asp 370 375 380 Thr LeuIle Glu Phe Tyr Ala Pro Trp Cys Gly His Cys Lys Lys Leu 385 390 395 400Thr Pro Ile Tyr Glu Glu Leu Ala Gln Lys Leu Gln Asp Glu Asp Val 405 410415 Ala Ile Val Lys Met Asp Ala Thr Ala Asn Asp Val Pro Pro Glu Phe 420425 430 Asn Val Arg Gly Phe Pro Thr Leu Phe Trp Leu Pro Lys Asp Ala Lys435 440 445 Asn Lys Pro Val Ser Tyr Asn Gly Gly Arg Glu Val Asp Asp PheLeu 450 455 460 Lys Tyr Ile Ala Lys Glu Ala Thr Thr Glu Leu Lys Gly PheAsp Arg 465 470 475 480 Ser Gly Lys Pro Lys Lys Thr Glu Leu 485 <210>SEQ ID NO 30 <211> LENGTH: 508 <212> TYPE: PRT <213> ORGANISM: Homosapiens <400> SEQUENCE: 30 Met Leu Arg Arg Ala Leu Leu Cys Leu Ala ValAla Ala Leu Val Arg 1 5 10 15 Ala Asp Ala Pro Glu Glu Glu Asp His ValLeu Val Leu Arg Lys Ser 20 25 30 Asn Phe Ala Glu Ala Leu Ala Ala His LysTyr Leu Leu Val Glu Phe 35 40 45 Tyr Ala Pro Trp Cys Gly His Cys Lys AlaLeu Ala Pro Glu Tyr Ala 50 55 60 Lys Ala Ala Gly Lys Leu Lys Ala Glu GlySer Glu Ile Arg Leu Ala 65 70 75 80 Lys Val Asp Ala Thr Glu Glu Ser AspLeu Ala Gln Gln Tyr Gly Val 85 90 95 Arg Gly Tyr Pro Thr Ile Lys Phe PheArg Asn Gly Asp Thr Ala Ser 100 105 110 Pro Lys Glu Tyr Thr Ala Gly ArgGlu Ala Asp Asp Ile Val Asn Trp 115 120 125 Leu Lys Lys Arg Thr Gly ProAla Ala Thr Thr Leu Pro Asp Gly Ala 130 135 140 Ala Ala Glu Ser Leu ValGlu Ser Ser Glu Val Ala Val Ile Gly Phe 145 150 155 160 Phe Lys Asp ValGlu Ser Asp Ser Ala Lys Gln Phe Leu Gln Ala Ala 165 170 175 Glu Ala IleAsp Asp Ile Pro Phe Gly Ile Thr Ser Asn Ser Asp Val 180 185 190 Phe SerLys Tyr Gln Leu Asp Lys Asp Gly Val Val Leu Phe Lys Lys 195 200 205 PheAsp Glu Gly Arg Asn Asn Phe Glu Gly Glu Val Thr Lys Glu Asn 210 215 220Leu Leu Asp Phe Ile Lys His Asn Gln Leu Pro Leu Val Ile Glu Phe 225 230235 240 Thr Glu Gln Thr Ala Pro Lys Ile Phe Gly Gly Glu Ile Lys Thr His245 250 255 Ile Leu Leu Phe Leu Pro Lys Ser Val Ser Asp Tyr Asp Gly LysLeu 260 265 270 Ser Asn Phe Lys Thr Ala Ala Glu Ser Phe Lys Gly Lys IleLeu Phe 275 280 285 Ile Phe Ile Asp Ser Asp His Thr Asp Asn Gln Arg IleLeu Glu Phe 290 295 300 Phe Gly Leu Lys Lys Glu Glu Cys Pro Ala Val ArgLeu Ile Thr Leu 305 310 315 320 Glu Glu Glu Met Thr Lys Tyr Lys Pro GluSer Glu Glu Leu Thr Ala 325 330 335 Glu Arg Ile Thr Glu Phe Cys His ArgPhe Leu Glu Gly Lys Ile Lys 340 345 350 Pro His Leu Met Ser Gln Glu LeuPro Glu Asp Trp Asp Lys Gln Pro 355 360 365 Val Lys Val Leu Val Gly LysAsn Phe Glu Asp Val Ala Phe Asp Glu 370 375 380 Lys Lys Asn Val Phe ValGlu Phe Tyr Ala Pro Trp Cys Gly His Cys 385 390 395 400 Lys Gln Leu AlaPro Ile Trp Asp Lys Leu Gly Glu Thr Tyr Lys Asp 405 410 415 His Glu AsnIle Val Ile Ala Lys Met Asp Ser Thr Ala Asn Glu Val 420 425 430 Glu AlaVal Lys Val His Ser Phe Pro Thr Leu Lys Phe Phe Pro Ala 435 440 445 SerAla Asp Arg Thr Val Ile Asp Tyr Asn Gly Glu Arg Thr Leu Asp 450 455 460Gly Phe Lys Lys Phe Leu Glu Ser Gly Gly Gln Asp Gly Ala Gly Asp 465 470475 480 Asp Asp Asp Leu Glu Asp Leu Glu Glu Ala Glu Glu Pro Asp Met Glu485 490 495 Glu Asp Asp Asp Gln Lys Ala Val Lys Asp Glu Leu 500 505<210> SEQ ID NO 31 <211> LENGTH: 364 <212> TYPE: PRT <213> ORGANISM:Medicago sativa <400> SEQUENCE: 31 Met Lys Met Glu Met His Gln Ile TrpSer Arg Ile Ala Leu Ala Ser 1 5 10 15 Phe Ala Phe Ala Ile Leu Phe ValSer Val Ser Ala Asp Asp Val Val 20 25 30 Val Leu Thr Glu Glu Asn Phe GluLys Glu Val Gly His Asp Lys Gly 35 40 45 Ala Leu Val Glu Phe Tyr Ala ProTrp Cys Gly His Cys Lys Lys Leu 50 55 60 Ala Pro Glu Tyr Glu Lys Leu ProAsn Ser Phe Lys Lys Ala Lys Ser 65 70 75 80 Val Leu Ile Ala Lys Val AspCys Asp Glu His Lys Ser Val Cys Ser 85 90 95 Lys Tyr Gly Val Ser Gly TyrPro Thr Ile Gln Trp Phe Pro Lys Gly 100 105 110 Ser Leu Glu Pro Lys LysPhe Glu Gly Pro Arg Thr Ala Glu Ser Leu 115 120 125 Ala Glu Phe Val AsnThr Glu Gly Gly Thr Asn Val Lys Ile Ala Thr 130 135 140 Ala Pro Ser HisVal Val Val Leu Thr Pro Glu Thr Phe Asn Glu Val 145 150 155 160 Val LeuAsp Gly Thr Lys Asp Val Leu Val Glu Phe Tyr Ala Pro Trp 165 170 175 CysGly His Cys Lys Ser Leu Ala Pro Ile Tyr Glu Lys Val Ala Ala 180 185 190Val Phe Lys Ser Glu Asp Asp Val Val Ile Ala Asn Leu Asp Ala Asp 195 200205 Lys Tyr Arg Asp Leu Ala Glu Lys Tyr Asp Val Ser Gly Phe Pro Thr 210215 220 Leu Lys Phe Phe Pro Lys Gly Asn Lys Ala Gly Glu Asp Tyr Gly Gly225 230 235 240 Gly Arg Asp Leu Asp Asp Phe Val Ala Phe Ile Asn Glu LysSer Gly 245 250 255 Thr Ser Arg Asp Ala Lys Gly Gln Leu Thr Ser Glu AlaGly Ile Val 260 265 270 Glu Asp Leu Asp Glu Leu Val Lys Glu Phe Val AlaAla Asn Asp Glu 275 280 285 Glu Lys Lys Ala Val Phe Ala Arg Ile Glu GluGlu Val Lys Lys Leu 290 295 300 Glu Gly Ser Ala Ser Arg Tyr Gly Lys IleTyr Leu Lys Val Ser Lys 305 310 315 320 Lys Tyr Leu Glu Lys Gly Ser AspTyr Ala Lys Asn Glu Ile Gln Arg 325 330 335 Leu Glu Arg Leu Leu Glu LysSer Ile Ser Pro Ala Lys Ala Asp Glu 340 345 350 Leu Thr Leu Lys Lys AsnIle Leu Ser Thr Tyr Ala 355 360 <210> SEQ ID NO 32 <211> LENGTH: 433<212> TYPE: PRT <213> ORGANISM: Drosophila melanogaster <400> SEQUENCE:32 Met Arg Gln Leu Ala Ser Ile Leu Leu Leu Ala Phe Val Val Gly Ser 1 510 15 Val Ser Ala Phe Tyr Ser Pro Ser Asp Gly Val Val Glu Leu Thr Pro 2025 30 Ser Asn Phe Asp Arg Glu Val Leu Lys Asp Asp Ala Ile Trp Val Val 3540 45 Glu Phe Tyr Ala Pro Trp Cys Gly His Cys Gln Ser Leu Val Pro Glu 5055 60 Tyr Lys Lys Leu Ala Lys Ala Leu Lys Gly Val Val Lys Val Gly Ser 6570 75 80 Val Asn Ala Asp Ala Asp Ser Thr Leu Ser Gly Gln Phe Gly Val Arg85 90 95 Gly Phe Pro Thr Ile Lys Ile Phe Gly Ala Asn Lys Lys Ser Pro Thr100 105 110 Asp Tyr Asn Gly Gln Arg Thr Ala Lys Ala Ile Ala Glu Ala AlaLeu 115 120 125 Ala Glu Val Lys Lys Lys Val Gln Gly Val Leu Gly Gly GlyGly Gly 130 135 140 Ser Ser Ser Gly Gly Ser Gly Ser Ser Ser Gly Asp AspVal Ile Glu 145 150 155 160 Leu Thr Glu Asp Asn Phe Asp Lys Leu Val LeuAsn Ser Asp Asp Ile 165 170 175 Trp Leu Val Glu Phe Phe Ala Pro Trp CysGly His Cys Lys Asn Leu 180 185 190 Ala Pro Glu Trp Ala Lys Ala Ala LysGlu Leu Lys Gly Lys Val Lys 195 200 205 Leu Gly Ala Leu Asp Ala Thr AlaHis Gln Ser Lys Ala Ala Glu Tyr 210 215 220 Asn Val Arg Gly Tyr Pro ThrIle Lys Phe Phe Pro Ala Gly Ser Lys 225 230 235 240 Arg Ala Ser Asp AlaGln Glu Tyr Asp Gly Gly Arg Thr Ala Ser Asp 245 250 255 Ile Val Ser TrpAla Ser Asp Lys His Val Ala Asn Val Pro Ala Pro 260 265 270 Glu Leu IleGlu Ile Ile Asn Glu Ser Thr Phe Glu Thr Ala Cys Glu 275 280 285 Gly LysPro Leu Cys Val Val Ser Val Leu Pro His Ile Leu Asp Cys 290 295 300 AspAla Lys Cys Arg Asn Lys Phe Leu Asp Thr Leu Arg Thr Leu Gly 305 310 315320 Glu Lys Phe Lys Gln Lys Gln Trp Gly Trp Ala Trp Ala Glu Gly Gly 325330 335 Gln Gln Leu Ala Leu Glu Glu Ser Leu Glu Val Gly Gly Phe Gly Tyr340 345 350 Pro Ala Met Ala Val Val Asn Phe Lys Lys Met Lys Phe Ser ValLeu 355 360 365 Lys Gly Ser Phe Ser Lys Asp Gly Ile Asn Glu Phe Leu ArgAsp Ile 370 375 380 Ser Tyr Gly Arg Gly His Thr Ala Pro Val Arg Gly AlaLys Lys Pro 385 390 395 400 Ala Ile Val Ser Val Asp Pro Trp Asp Gly LysAsp Gly Gln Leu Pro 405 410 415 Thr Glu Glu Asp Ile Asp Leu Ser Asp IleAsp Leu Asp Lys Asp Glu 420 425 430 Leu

What is claimed is:
 1. An isolated polynucleotide comprising: (a) anucleic acid sequence encoding a polypeptide having protein disulfideisomerase activity, wherein the polypeptide has an amino acid sequenceof at least 80% sequence identity based on Clustal method of alignmentwhen compared to one of SEQ ID NO:16 or 18; or (b) a complement of thenucleic acid sequence wherein the complement and nucleic acid sequenceconsist of the same number of nucleotides and are 100% complementary. 2.A chimeric gene comprising the isolated polynucleotide of claim 1operably linked to at least one regulatory sequence.
 3. A host cellcomprising the chimeric gene of claim
 2. 4. An isolated host cellcompromising an isolated polynucleotide of claim
 1. 5. The isolated hostcell of claim 4 wherein the isolated host is selected from the groupconsisting of plant, insect, yeast, bacteria, and virus.
 6. A viruscomprising the isolated polynucleotide of claim
 1. 7. The polynucleotideof claim 1, wherein the amino acid sequence of the polypeptide and theamino acid sequence of SEQ ID NO:16 or 18 have at least 90% identitybased on the Clustal alignment method.
 8. The polynucleotide of claim 1,wherein the amino acid sequence of the polypeptide and the amino acidsequence of SEQ ID NO:16 or 18 have at least 95% identity based on theClustal alignment method.
 9. The polynucleotide of claim 1, wherein thenucleic acid sequence comprises the nucleotide sequence of SEQ ID NO:15or
 17. 10. The polynucleotide of claim 1, wherein the amino acidsequence of the polypeptide comprises the amino acid sequence of SEQ IDNO:16 or
 18. 11. A vector comprising the polynucleotide of claim
 1. 12.A method for transforming a cell comprising transforming a cell with thepolynucleotide of claim
 1. 13. A recombinant baculovirus comprising thepolynucleotide of claim
 1. 14. A method for producing a plant comprisingtransforming a plant cell with the polynucleotide of claim 1 andregenerating a plant from the transformed plant cell.
 15. A method forenhancing the yield of a transgenic secreted protein by co-transforminga cell with a polynucleotide encoding a secreted polypeptide and apolynucleotide of claim
 1. 16. A method for isolating a polypeptideencoded by the polynucleotide of claim 1 comprising isolating thepolypeptide from a cell containing a chimeric gene comprising thepolynucleotide operably linked to a regulatory sequence.